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Research & Scholarship

Current Research and Scholarly Interests

Our research concentrates on the study of malignant lymphoma and tumors of the immune system using the tools of immunology and molecular biology to develop a better understanding of the initiation and progression of the malignant process. Receptor molecules present on the surface of tumor cells transmit signals for regulation of cell growth. These receptors include the immunoglobulin molecule on B cell tumors and the T cell receptor on T cell tumors. Questions the lab is currently addressing include:1. Can a clue to the pathogenesis of lymphoma be derived from a study of their antigen receptors?2. Can new treatments for lymphoma be developed by targeting receptors with monoclonal antibodies?3. Can vaccines be developed which can induce an immune response in the host against the receptors on their own tumor?

Clinical Trials

Clinical and Pathologic Studies in Non-Hodgkin's Lymphoma and Hodgkin's DiseaseRecruiting

The purpose of this study is to characterize the molecular and cell biology of the tumor
cells in lymphoma.

For patients with lymphoma that recurs after chemotherapy, bone marrow transplantation using
cells from a healthy donor represents potentially curative treatment. In these individuals,
cure is possible because transplantation of healthy donor immune cells can fight the
lymphoma in the patient. The goal of this work is to test a strategy that activates the
healthy donor immune cells so that they more effectively fight lymphoma and can result in an
increased cure rate for these patients. Our group has previously studied CpG, an immune
activating medication, in patients with lymphoma and demonstrated modest anti-tumor
responses. We now have a more potent form of CpG which we intend to test to see if it will
better activate the donor immune cells and result in shrinkage of tumor throughout the
entire body, not just at the injected site.

Stanford is currently not accepting patients for this trial.For more information, please contact Physician Referrals, 650-723-0822.

Phase I/II Study of Intratumoral Injection of CPG 7909, a TLR9 Agonist, Combined With Local Radiation for Patients With Recurrent Mycosis Fungoides.Not Recruiting

This is a single institution phase I / II trial to evaluate the safety and efficacy of
intratumoral CpG injections combined with local radiation in patients with mycosis
fungoides. Patients will receive low-dose radiotherapy to a single tumor site on days 1 and
2 (2 Gy each day). CpG injections will be administered into the same tumor site within 24
hours before or 24 hours after each radiation treatment. Weekly doses of (intratumoral or
peritumoral injections) CpG will be then administered subcutaneously in the region of
previous injections for 23 additional doses. The total treatment duration is 24 weeks.

Stanford is currently not accepting patients for this trial.For more information, please contact Mayita Romero, (650) 725 - 6452.

This study is to determine the safety and effectiveness of VTX-2337 (an investigational drug
that stimulates the immune system) in combination with radiation therapy in treating
patients with low-grade B-cell lymphoma. Patients will receive 2 low doses of radiotherapy,
and 9 intratumoral injections of VTX-2337 over the course of 3 months.

Stanford is currently not accepting patients for this trial.For more information, please contact Lori Richards, (650) 725 - 8589.

This Phase 1/2, open-label, single-arm, efficacy, safety, and pharmacodynamic study is to
evaluate safety and efficacy of idelalisib (GS-1101, CAL-101) in patients with previously
treated indolent non-Hodgkin lymphoma (iNHL).
Eligible patients will initiate oral therapy with idelalisib at a starting dose of 150 mg
twice per day. Treatment with idelalisib can continue in compliant participants for up to
twelve 28-day cycles of idelalisib. Participants who appear to be benefiting from treatment
at the completion of 12 cycles of treatment with idelalisib may be eligible for
participation in a long-term safety extension study of idelalisib.

Stanford is currently not accepting patients for this trial.For more information, please contact Lori Richards, (650) 725 - 4968.

Personalized Active Immunotherapy (Vaccine Therapy) and Sargramostim Given After Standard of Care Treatment With Rituximab and Chemotherapy for Initial Treatment With LymphomaNot Recruiting

The treatment being investigated is a patient- and tumor-specific therapy known as a
personalized active immunotherapy. Personalized active immunotherapy is an attempt to use a
person's own immune system to combat disease. Sargramostim (a.k.a. GM-CSF) is given together
with the personalized active immunotherapy because it may increase the immune system's
response and, therefore, aid in the effect of the personalized active immunotherapy.
This approach has previously been studied in patients with follicular Non-Hodgkin's lymphoma
and other B-cell malignancies. Encouraging efficacy results and a favorable safety profile
have been seen to date in these studies.

Stanford is currently not accepting patients for this trial.For more information, please contact Susie Bawn, (650) 725 - 4968.

The purpose of the study is to assess the safety, tolerability, pharmacokinetics and
immunoregulatory activity of urelumab (BMS-663513) in cancer subjects with advanced and/or
metastatic tumors and relapsed/refractory B-Cell Non-Hodgkin's Lymphoma

Stanford is currently not accepting patients for this trial.For more information, please contact Ami Okada, 650-725-4968.

The purpose of this study is to better understand the biology of the body's immune response
to monoclonal antibody therapy for cancer. Your health information will be used to identify
your tissues. The tissue we obtain may be useful for research or education, resulting in new
drugs, therapies or diagnostic procedures.

This pilot phase I/II trial studies the side effects and best of dose ipilimumab when given
together with local radiation therapy and to see how well it works in treating patients with
recurrent melanoma, non-Hodgkin lymphoma, colon, or rectal cancer. Monoclonal antibodies,
such as ipilimumab, can block cancer growth in different ways. Some block the ability of
cancer cells to grow and spread. Others find cancer cells and help kill them or carry
cancer-killing substances to them. Radiation therapy uses high energy x rays to kill cancer
cells. Giving monoclonal antibody therapy together with radiation therapy may be an
effective treatment for melanoma, non-Hodgkin lymphoma, colon, or rectal cancer

Stanford is currently not accepting patients for this trial.For more information, please contact Erin Waller, 650-725-0379.

Genes in Predicting Outcome of Patients With DLBCL Treated With Rituximab and Combination Chemotherapy (R-CHOP)Not Recruiting

The investigators hypothesize that survival of newly diagnosed DLBCL (diffuse large B-cell
lymphoma) patients treated with R-CHOP can be predicted by RNA or protein gene expression or
by presence of biomarkers associated with the anti-tumor effects of Rituximab.

We hope to learn whether I-131 tositumomab combined with external beam radiation therapy is
an effective means of treating relapsed, bulky non-Hodgkin's lymphoma. The purpose of the
study is to determine the overall response rate with responses described as: Site-dependent
and overall CR and functional CR (CR of CRu(Complete Response Unconfirmed)/PR with PET
negativity), or PR rates.

Stanford is currently not accepting patients for this trial.For more information, please contact Lucy Schoen, (650) 725 - 1718.

Phase 1-2 of a CpG-Activated Whole Cell Vaccine Followed by Autologous Immunotransplant for MCLNot Recruiting

Mantle Cell Lymphoma is a sub-type of Non-Hodgkin's Lymphoma which is generally considered
incurable with current therapy. Our goal is to accrue 59 patients who receive an autologous
vaccine against their individual lymphoma after undergoing stem cell transplantation. Our
hope is that vaccination will prolong the time which patients will stay in remission from
their disease.

Stanford is currently not accepting patients for this trial.For more information, please contact Ami Okada, (650) 725 - 4968.

Patients meeting specific inclusion and exclusion criteria will be enrolled in two stages,
19 patients in Stage 1 and 36 patients in Stage 2. Stage 2 will enroll if 4 or more patients
exhibit a response at Week 8 or later in the study. All enrolled patients will be treated
with Fostamatinib Disodium until disease progression. Efficacy will be assessed by tumor
measurements using CT and PET (when indicated) scans and physical exam at baseline, and
scans and physical exam of all disease-involved areas every 8 weeks until progression.
Safety will be assessed by periodic physical exams, clinical laboratory studies, and adverse
events. All patients will have a follow-up visit 30 days following last study drug
treatment. Blood samples for PK assessment will be obtained from all patients enrolled in
Stage 1 at protocol defined intervals.

Stanford is currently not accepting patients for this trial.For more information, please contact Sipra Choudhury, (650) 736 - 2563.

A Study Of PF-05082566 As A Single Agent And In Combination With RituximabNot Recruiting

A study of PF-05082566, a 4-1BB agonist monoclonal antibody (mAb), in patients with solid
tumors or b-cell lymphomas, and in combination with rituximab in patients with CD20 positive
Non-Hodgkin's Lymphoma (NHL).

Stanford is currently not accepting patients for this trial.For more information, please contact Ami Okada, (650) 725 - 4968.

RATIONALE: Vaccines made from a person's cancer cells may make the body build an effective
immune response to kill cancer cells. Colony-stimulating factors, such as GM-CSF, may
increase the number of immune cells found in bone marrow or peripheral blood and may
stimulate the immune system in different ways and stop cancer cells from growing.
PURPOSE: This phase II trial is studying how well giving vaccine therapy together with
GM-CSF works in treating patients with progressive B-cell non-Hodgkin's lymphoma.

Stanford is currently not accepting patients for this trial.For more information, please contact Mayita Romero, (650) 725 - 6452.

Publications

All Publications

Abstract

Systemic administration of the checkpoint blockade antibody anti-CTLA4 results in severe autoimmune toxicity, limiting its clinical efficacy. Fransen and colleagues show here that peritumoral delivery of low doses of this immunomodulatory drug can trigger a systemic antitumor immune response while preventing the toxicity against other organs.

Abstract

Recent studies report an improvement in overall survival (OS) of patients with follicular lymphoma (FL). Previously untreated patients with grade 1-2 FL referred from 1960-2003 and treated at Stanford were identified. Four eras were considered: era 1, pre-anthracycline (1960-1975, n=180); era 2, anthracycline (1976-1986, n=426), era 3, aggressive chemotherapy/purine analogs (1987-1996, n=471) and era 4, rituximab (1997-2003, n=257). Clinical characteristics, patterns of care and survival outcomes were assessed. Observed OS was compared with the expected OS calculated from Berkeley Mortality Database life tables derived from population matched by gender and age at time of diagnosis. The median OS was 13.6 years. Age, gender and stage did not differ across the eras. Although primary treatment varied, event free survival after the first treatment did not differ between eras (p=0.17). Median OS improved from approximately 11 years in eras 1 and 2 to 18.4 years in era 3 and has not yet been reached for era 4 (p<0.001) with no suggestion of a plateau in any era. These improvements in OS exceeded improvements in survival in the general population during the same time period. Several factors, including better supportive care and effective therapies for relapsed disease, are likely responsible for this improvement.

Abstract

Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies.

Abstract

Defects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4-, IL-10-, and IL-21-induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein-specific flow cytometry with several T-cell markers, we identified that CD4(+)CD45RO(+)CD62L(-) FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4(+)PD-1(hi) FL TILs, containing T(FH) and non-T(FH) cells, had lost their cytokine responsiveness, whereas PD-1 TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1(+) histiocytes were found within the T cell-rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1(hi) subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.

Abstract

Follicular lymphoma is a monoclonal B-cell malignancy with each patient's tumor expressing a unique cell surface immunoglobulin (Ig), or B-cell receptor (BCR), that can potentially recognize antigens and/or transduce signals into the tumor cell. Here we evaluated the reactivity of tumor derived Igs for human tissue antigens. Self-reactivity was observed in 26% of tumor Igs (25 of 98). For one follicular lymphoma patient, the recognized self-antigen was identified as myoferlin. This patient's tumor cells bound recombinant myoferlin in proportion to their level of BCR expression, and the binding to myoferlin was preserved despite ongoing somatic hypermutation of Ig variable regions. Furthermore, BCR-mediated signaling was induced after culture of tumor cells with myoferlin. These results suggest that antigen stimulation may provide survival signals to tumor cells and that there is a selective pressure to preserve antigen recognition as the tumor evolves.

Abstract

Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (?CD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the ?CD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the ?CD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.

Abstract

CD137 (also known as 4-1BB and TNFRSF9) is a member of the tumor necrosis factor receptor superfamily. Originally identified as a costimulatory molecule expressed by activated T cells and NK cells, CD137 is also expressed by follicular dendritic cells, monocytes, mast cells, granulocytes, and endothelial cells. Anti-CD137 immunotherapy has recently shown promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. We defined the expression of CD137 protein in both normal and neoplastic hematolymphoid tissue. CD137 protein is expressed by follicular dendritic cells in the germinal center and scattered paracortical T cells, but not by normal germinal-center B cells, bone marrow progenitor cells, or maturing thymocytes. CD137 protein is expressed by a select group of hematolymphoid tumors, including classical Hodgkin lymphoma, T-cell and NK/T-cell lymphomas, and follicular dendritic cells neoplasms. CD137 is a novel diagnostic marker of these tumors and suggests a possible target for tumor-directed antibody therapy.

Abstract

The clinical efficacy of monoclonal antibodies as cancer therapeutics is largely dependent upon their ability to target the tumor and induce a functional antitumor immune response. This two-step process of ADCC utilizes the response of innate immune cells to provide antitumor cytotoxicity triggered by the interaction of the Fc portion of the antibody with the Fc receptor on the immune cell. Immunotherapeutics that target NK cells, ?? T cells, macrophages and dendritic cells can, by augmenting the function of the immune response, enhance the antitumor activity of the antibodies. Advantages of such combination strategies include: the application to multiple existing antibodies (even across multiple diseases), the feasibility (from a regulatory perspective) of combining with previously approved agents and the assurance (to physicians and trial participants) that one of the ingredients - the antitumor antibody - has proven efficacy on its own. Here we discuss current strategies, including biologic rationale and clinical results, which enhance ADCC in the following ways: strategies that increase total target-monoclonal antibody-effector binding, strategies that trigger effector cell 'activating' signals and strategies that block effector cell 'inhibitory' signals.

Abstract

Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (T(reg)). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding T(regs). In a murine model of B-cell lymphoma, only CD137(neg)CD44(hi) CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137(pos)CD44hi CD4 T cells consisted primarily of activated T(regs). Notably, this CD137(pos) T(reg) population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137(pos) cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137(pos)CD44(hi) CD4 cells to CD137(neg)CD44(hi) CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated T(regs) that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137(pos) CD4 T cells that were predominantly CD25(pos)FoxP3(pos) T(regs). In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory T(reg) population.

Complementary costimulation of human T-cell subpopulations by cluster of differentiation 28 (CD28) and CD81PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASagi, Y., Landrigan, A., Levy, R., Levy, S.2012; 109 (5): 1613-1618

Abstract

Cluster of differentiation 81 (CD81) is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells: Primary human naïve T cells are better costimulated by CD81, whereas the memory T-cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared with that by CD28. We found that IL-6 played a role in the activation of the naïve T-cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T-cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T-cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire.

Abstract

Monoclonal antibodies (mAbs) are in widespread use for the treatment of cancer. Their success as cancer therapeutics relies substantially on their ability to engage the immune system. Specifically, Fc-receptor-expressing immune cells mediate the killing of tumor cells by mAbs. Stimulation of these immune effector cells might therefore represent a promising strategy to enhance the therapeutic potential of mAbs. For instance, stimulation of natural killer cells, ?? T cells, macrophages, or dendritic cells can be used to enhance antibody-dependent cellular cytotoxicity, phagocytosis or even tumor vaccine effects. Here, we review several ways to improve the antitumor efficacy of mAbs by combining them with therapies that are directed against immune effector cells.

Abstract

Several gene-expression signatures predict survival in diffuse large B-cell lymphoma (DLBCL), but the lack of practical methods for genome-scale analysis has limited translation to clinical practice. We built and validated a simple model using one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). LIM domain only 2 (LMO2) was validated as an independent predictor of survival and the "germinal center B cell-like" subtype. Expression of tumor necrosis factor receptor superfamily member 9 (TNFRSF9) from the DLBCL microenvironment was the best gene in bivariate combination with LMO2. Study of TNFRSF9 tissue expression in 95 patients with DLBCL showed expression limited to infiltrating T cells. A model integrating these 2 genes was independent of "cell-of-origin" classification, "stromal signatures," IPI, and added to the predictive power of the IPI. A composite score integrating these genes with IPI performed well in 3 independent cohorts of 545 DLBCL patients, as well as in a simple assay of routine formalin-fixed specimens from a new validation cohort of 147 patients with DLBCL. We conclude that the measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL.

Abstract

Conventional chemotherapy for lymphoma has advanced greatly over the past 50 years, changing some lymphoma subtypes from uniformly lethal to curable; however, the majority of lymphomas in patients remain incurable, and there is a need for novel therapies with less toxicity and more specific targeting of tumor cells. The vertebrate immune system has evolved the capacity for such specific targeting through the B-cell and T-cell receptors; passive immunotherapies utilizing these receptors, such as monoclonal antibodies (mAbs) or T cells, have shown efficacy in treating lymphomas. The first generation of mAb-based therapies has transformed the standard of care for lymphoma, and newer antibodies may improve on this approach. Clinical activity has been shown by T cells bearing receptors that target viral antigens as well as T cells bearing re-engineered receptors that target antigens recognized by antibodies. Active immunotherapies, such as vaccines and immune checkpoint blockades, have prolonged survival in certain solid tumors and are being actively pursued to treat lymphoma. A variety of vaccines (eg, protein- and cell-based vaccines) are being tested in ongoing trials, and the most recent iterations show therapeutic activity. Newer trials are addressing the problem of tumor-induced immunosuppression by the use of antibodies against immunologic checkpoints or by the reinfusion of primed T cells after lymphodepletion, a process we refer to as immunotransplantation. Herein, we discuss results of the various immunotherapy strategies applied to lymphoma and the ongoing approaches for their improvement.

Abstract

We studied the efficacy of 2 germinal center B-cell markers, HGAL and LMO2, in the separation of lymphomas derived from small B cells, particularly follicular lymphoma (FL) and marginal zone lymphoma occurring in nodal, extranodal, splenic, and bone marrow sites using immunohistochemical analysis for CD10, BCL6, BCL2, HGAL, and LMO2. Our results showed that HGAL and LMO2 are sensitive and specific markers for detecting FL in nodal and extranodal sites. In contrast, all markers were down-regulated in FL infiltrates in the bone marrow. CD10 and HGAL were expressed in a subset of FLs in the bone marrow and were highly correlated with each other and with CD21, a marker of follicular dendritic cells. We conclude that HGAL and LMO2 should be considered in immunohistochemical panels used for the routine workup of lymphomas derived from small B cells. In the bone marrow, staining for HGAL or CD10 can be helpful in making a diagnosis of FL, although they are absent in a subset of cases.

Abstract

The aim of cancer immunotherapy is to induce immune cells to kill tumor and promote immunological memory that protects against tumor recurrence. Most current immunotherapies, such as monoclonal antibodies (mAb), target the tumor cells directly. Advances in our understanding of the immune system such as the role of co-stimulatory and co-inhibitory receptors, and the advent of new immunomodulatory agents provide new opportunities to target the immune system and enhance anti-tumor immune responses. These promising agents include immunomodulating mAbs, Toll-like receptor agonists, IMiDs, and cytokines. In this review, we discuss the current results of immunomodulating agents in the treatment of hematological malignancies and propose applications that include targeting of the innate and adaptive immune systems as well as combinations with tumor-specific mAbs.

Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.

Abstract

We designed a whole tumor cell vaccine by "loading" lymphoma tumor cells with CG-enriched oligodeoxynucleotide (CpG), a ligand for the Toll-like receptor 9 (TLR9). CpG-loaded tumor cells were phagocytosed, delivering both tumor antigen(s) and the immunostimulatory CpG molecule to antigen-presenting cells (APCs). These APCs then expressed increased levels of costimulatory molecules and induced T-cell immunity. TLR9 was required in the APCs but not in the CpG-loaded tumor cell. We demonstrate that T cells induced by this vaccine are effective in adoptive cellular therapy for lymphoma. T cells from vaccinated mice transferred into irradiated, syngeneic recipients protected against subsequent lymphoma challenge and, remarkably, led to regression of large and established tumors. This therapeutic effect could be transferred by CD4(+) but not by CD8(+) T cells. A CpG-loaded whole-cell vaccination is practical and has strong potential for translation to the clinical setting. It is currently being tested in a clinical trial of adoptive immunotherapy for mantle-cell lymphoma.

Abstract

The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment - TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.

Abstract

Combining tumor antigens with an immunostimulant can induce the immune system to specifically eliminate cancer cells. Generally, this combination is accomplished in an ex vivo, customized manner. In a preclinical lymphoma model, intratumoral injection of a Toll-like receptor 9 (TLR9) agonist induced systemic antitumor immunity and cured large, disseminated tumors.We treated 15 patients with low-grade B-cell lymphoma using low-dose radiotherapy to a single tumor site and-at that same site-injected the C-G enriched, synthetic oligodeoxynucleotide (also referred to as CpG) TLR9 agonist PF-3512676. Clinical responses were assessed at distant, untreated tumor sites. Immune responses were evaluated by measuring T-cell activation after in vitro restimulation with autologous tumor cells.This in situ vaccination maneuver was well-tolerated with only grade 1 to 2 local or systemic reactions and no treatment-limiting adverse events. One patient had a complete clinical response, three others had partial responses, and two patients had stable but continually regressing disease for periods significantly longer than that achieved with prior therapies. Vaccination induced tumor-reactive memory CD8 T cells. Some patients' tumors were able to induce a suppressive, regulatory phenotype in autologous T cells in vitro; these patients tended to have a shorter time to disease progression. One clinically responding patient received a second course of vaccination after relapse resulting in a second, more rapid clinical response.In situ tumor vaccination with a TLR9 agonist induces systemic antilymphoma clinical responses. This maneuver is clinically feasible and does not require the production of a customized vaccine product.

Abstract

Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

Abstract

Follicular lymphoma (FL) can exhibit variant histologic patterns that can lead to confusion with other B-cell lymphomas and reactive conditions. Diagnostic markers such as CD10 and BCL2 may be difficult to interpret in variant FL patterns, and are often diminished or absent in the interfollicular and diffuse components. We evaluated 2 recently characterized germinal center B-cell markers, human germinal center associated lymphoma (HGAL), and LIM-only transcription factor 2 (LMO2), in 127 FL patient biopsies (94 nodal, 33 extranodal), and correlated the findings with histologic pattern, cellular composition, grade, and additional immunostains (CD20, CD3, CD21, CD10, BCL2, and BCL6). Architectural patterns included predominantly follicular (75%) and follicular and diffuse components (25%); 10 cases showed marginal zone differentiation and 3 were floral variants. Eighty-nine cases were low grade (38 grade 1; 51 grade 2) and 38 were grade 3 (29 grade 3A and 9 grade 3B). HGAL had the highest overall sensitivity of detecting FL and was superior in detecting the interfollicular and diffuse components compared with BCL2, LMO2, CD10, and BCL6. All 28 cases that lacked CD10, expressed HGAL, and the majority also expressed LMO2. Our results show that HGAL and LMO2 are sensitive markers for FL diagnosis. The addition of HGAL and LMO2 to the immunohistologic panel is beneficial in the work-up of nodal and extranodal B-cell lymphomas and the efficacy of HGAL in detecting the follicular, interfollicular and diffuse components of FL is of particular value in the setting of variant immunoarchitectural patterns.

Abstract

Human tumors contain populations of both cancerous and host immune cells whose malignant signaling interactions may define each patient's disease trajectory. We used multiplexed phospho-flow cytometry to profile single cells within human follicular lymphoma tumors and discovered a subpopulation of lymphoma cells with impaired B cell antigen receptor (BCR) signaling. The abundance of BCR-insensitive cells in each tumor negatively correlated with overall patient survival. These lymphoma negative prognostic (LNP) cells increased as tumors relapsed following chemotherapy. Loss of antigen receptor expression did not explain the absence of BCR signaling in LNP tumor cells, and other signaling responses were intact in these cells. Furthermore, BCR signaling responses could be reactivated in LNP cells, indicating that BCR signaling is not missing but rather specifically suppressed. LNP cells were also associated with changes to signaling interactions in the tumor microenvironment. Lower IL-7 signaling in tumor infiltrating T cells was observed in tumors with high LNP cell counts. The strength of signaling through T cell mediator of B cell function CD40 also stratified patient survival, particularly for those whose tumors contained few LNP cells. Thus, analysis of cell-cell interactions in heterogeneous primary tumors using signaling network profiles can identify and mechanistically define new populations of rare and clinically significant cells. Both the existence of these LNP cells and their aberrant signaling profiles provide targets for new therapies for follicular lymphoma.

Abstract

Chemokine receptor 1 (CCR1) is a G protein-coupled receptor that binds to members of the C-C chemokine family. Recently, CCL3 (MIP-1alpha), a high-affinity CCR1 ligand, was identified as part of a model that independently predicts survival in patients with diffuse large B-cell lymphoma (DLBCL). However, the role of chemokine signaling in the pathogenesis of human lymphomas is unclear. In normal human hematopoietic tissues, we found CCR1 expression in intraepithelial B cells of human tonsil and granulocytic/monocytic cells in the bone marrow. Immunohistochemical analysis of 944 cases of hematolymphoid neoplasia identified CCR1 expression in a subset of B- and T-cell lymphomas, plasma cell myeloma, acute myeloid leukemia, and classical Hodgkin lymphoma. CCR1 expression correlated with the non-germinal center subtype of DLBCL but did not predict overall survival in follicular lymphoma. These data suggest that CCR1 may be useful for lymphoma classification and support a role for chemokine signaling in the pathogenesis of hematolymphoid neoplasia.

Abstract

CD81 is a tetraspanin cell surface protein that regulates CD19 expression in B lymphocytes and enables hepatitis C virus infection of human cells. Immunohistologic analysis in normal hematopoietic tissue showed strong staining for CD81 in normal germinal center B cells, a cell type in which its increased expression has not been previously recognized. High-dimensional flow cytometry analysis of normal hematopoietic tissue confirmed that among B- and T-cell subsets, germinal center B cells showed the highest level of CD81 expression. In more than 800 neoplastic tissue samples, its expression was also found in most non-Hodgkin lymphomas. Staining for CD81 was rarely seen in multiple myeloma, Hodgkin lymphoma, or myeloid leukemia. In hierarchical cluster analysis of diffuse large B-cell lymphoma, staining for CD81 was most similar to other germinal center B cell-associated markers, particularly LMO2. By flow cytometry, CD81 was expressed in diffuse large B-cell lymphoma cells independent of the presence or absence of CD10, another germinal center B-cell marker. The detection of CD81 in routine biopsy samples and its differential expression in lymphoma subtypes, particularly diffuse large B-cell lymphoma, warrant further study to assess CD81 expression and its role in the risk stratification of patients with diffuse large B-cell lymphoma.

Abstract

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Abstract

We investigated the ability of CpG-oligodeoxynucleotide to generate an anti-tumor CD8+ T-cell immune response and to synergize with passive antibody therapy. For these studies, we generated an antibody against the idiotype on the A20 B-cell lymphoma line. This antibody caused the regression of established tumors, but ultimately the tumors relapsed. The escaping surface IgG-negative tumor cells were resistant to both antibody-dependent cellular cytotoxicity and signaling-induced cell death. Addition of intratumoral CpG to antibody therapy cured large established tumors and prevented the occurrence of tumor escapees. The failure of the combination therapy in mice deficient for CD8+ T cells demonstrates the critical role of CD8+ T cells in tumor eradication. When mice were inoculated with 2 tumors and treated systemically with antibody followed by intratumoral CpG in just one tumor, both tumors regressed, indicating that a systemic immune response was generated. Although antibody therapy can eliminate tumor cells bearing the target antigen, it frequently selects for antigen loss variants. However, when a poly-specific T-cell response was generated against the tumor by intratumoral CpG, even large established tumors were cured. Such an immune response can prevent the emergence of antibody selected tumor escapees and provide long-lasting tumor protection.

Abstract

Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma patients eventually relapse. Induction of an adaptive immune response may elicit active and long-lasting antitumor immunity, thereby preventing or delaying recurrence. Immunomodulating mAbs directed against immune cell targets can be used to enhance the immune response to achieve efficient antitumor immunity. Anti-CD137 agonistic mAb has demonstrated antitumor efficacy in various tumor models and has now entered clinical trials for the treatment of solid tumors. Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. We found that human primary lymphoma tumors are infiltrated with CD137+ T cells. We therefore hypothesized that lymphoma would be susceptible to treatment with anti-CD137 agonistic mAb. Using a mouse model, we demonstrate that anti-CD137 therapy has potent antilymphoma activity in vivo. The antitumor effect of anti-CD137 therapy was mediated by both natural killer (NK) and CD8 T cells and induced long-lasting immunity. Moreover, the antitumor activity of anti-CD137 mAb could be further enhanced by depletion of regulatory T cell (T(regs)). These results support the evaluation of anti-CD137 therapy in clinical trials for patients with lymphoma.

Abstract

Therapy for non-Hodgkin lymphoma has benefited greatly from basic science and clinical research such that chemotherapy and monoclonal antibody therapy have changed some lymphoma subtypes from uniformly lethal to curable, but the majority of lymphoma patients remain incurable. Novel therapies with less toxicity and more specific targeting of tumor cells are needed and immunotherapy is among the most promising of these. Recently completed randomized trials of idiotype vaccines and earlier-phase trials of other vaccine types have shown the ability to induce antitumor T cells and some clinical responses. More recently, trials of adoptive transfer of antitumor T cells have demonstrated techniques to increase the persistence and antitumor effect of these cells. Herein, we discuss lymphoma immunotherapy clinical trial results and what lessons can be taken to improve their effect, including the combination of vaccination and adoptive transfer in an approach we have dubbed 'immunotransplant'.

Abstract

Previous studies demonstrated that vaccination-induced tumor-specific immune response is associated with superior clinical outcome in patients with follicular lymphoma. Here, we investigated whether this positive correlation extends to overall survival (OS). We analyzed 91 untreated patients who received CVP chemotherapy (cyclophosphamide, vincristine, and prednisone) followed by idiotype vaccination. Idiotype proteins were produced either by the hybridoma method or by expression of recombinant idiotype-encoding sequences in mammalian or plant-based expression systems. We found that achieving a complete response/complete response unconfirmed (CR/CRu) to CVP and making an anti-idiotype antibody are 2 independent factors that each correlated with longer OS at 10 years (89% vs 68% with or without a CR/CRu, P = .024; 90% vs 69% with or without tumor-specific antibody production; P = .027). In the subset of patients who received hybridoma-generated vaccines, we found that anti-idiotype production was even more highly associated with superior OS (P < .002); this was the case even in patients with a partial response (PR) to CVP (P < .001).

Abstract

Therapeutic vaccines for lymphomas have been developed to induce active and long-lasting immune responses against lymphoma capable of eradicating the tumor. Most of these vaccines use the tumor B cell idiotype (the unique variable region of the surface immunoglobulin) as a tumor-specific antigen. The first human clinical trial for lymphoma vaccine was initiated 20 years ago. Along with several other phase I/II trials, it showed encouraging results which supported the initiation of three phase III trials. The results of these trials have recently been released (although not published yet) which failed to demonstrate a prolongation in progression-free survival following chemotherapy. Despite this disappointing result, a number of observations have accumulated over the years that suggest some clinical efficacy of lymphoma vaccines. Several strategies are being developed to improve these results that include optimization of antigen delivery and presentation as well as enhancement of anti-tumor T cell function. This review describes the clinical development of lymphoma vaccines and delineates advances, problems and prospects towards integration of this strategy in the therapeutic armamentarium for lymphoma.

Abstract

We have previously shown that intratumoral injection of CpG oligodeoxynucleotide plus systemic chemotherapy can induce a T-cell immune response against lymphoma and serve as a therapeutic vaccine to cure tumors in a murine model. Here, we demonstrate that antibody-mediated modulation of T cells increases the efficacy of CpG vaccination, thereby eliminating the need for chemotherapy. T-cell modulation was accomplished by targeting both effector and regulatory T-cell populations using systemic administration of monoclonal antibodies against OX40, CTLA4, GITR, and folate receptor 4 (FR4). Each of these antibodies enhanced the effect of intratumoral CpG. Some pairwise combinations of these antibodies potentiated T-cell modulation and further enhanced the efficacy of CpG vaccination. Specifically, the combination of anti-OX40 and anti-CTLA4 which enhance activation and block cell-intrinsic negative regulatory circuits in T cells, respectively, was especially potent. When combined with intratumoral CpG, it induced antitumor CD4 and CD8 T-cell immunity, cured large and systemic lymphoma tumors without chemotherapy, and provided long-lasting immunity against tumor rechallenge. Our results show that the combination of intratumoral CpG and immunomodulatory T-cell antibodies has promise for therapeutic vaccination against lymphoma. These reagents are becoming available for human clinical trials.

Abstract

The transcription factor LMO2 is involved in vascular and hematopoietic development and hematolymphoid neoplasia. We have demonstrated that LMO2 is expressed nearly ubiquitously in native and neoplastic vasculature, including lymphatics. LMO2 reactivity is otherwise virtually absent in nonhematolymphoid tissues except in breast myoepithelium, prostatic basal cells, and secretory phase endometrial glands. Vasculature is LMO2- in adult and fetal heart, brain of older adults, hepatic sinusoids, and hepatocellular carcinoma. LMO2 is uniformly expressed in benign vascular and lymphatic neoplasms and in most malignant vascular neoplasms with the exception of epithelioid vascular neoplasms of pleura and bone. Among nonvascular neoplasms, LMO2 reactivity is present in giant cell tumor of tendon sheath, juvenile xanthogranuloma, a subset of gastrointestinal stromal tumors, small round blue cell tumors, and myoepithelial-derived neoplasms. The restricted expression pattern, nuclear localization, and crisp staining of LMO2 in paraffin blocks make it an attractive candidate for the diagnostic immunohistochemistry laboratory.

Abstract

Ex vivo-expanded tumor-infiltrating lymphocytes infused into lymphodepleted recipients has clear antitumor efficacy. More practical sources of such antitumor lymphocytes would broaden the application of this approach. Previously, we described an in situ vaccination combining chemotherapy with intratumoral injection of CpG-enriched oligonucleotides, which induced T-cell immunity against established lymphoma. An ongoing clinical trial of this maneuver has demonstrated clinical responses in lymphoma patients. Here, we use this vaccine maneuver to generate immune cells for transfer into irradiated, syngeneic recipients. Transferred tumor-specific T-effector (T(eff)) cells preferentially expanded, increasing the T(eff)/T-regulatory (T(reg)) ratio in these "immunotransplantation" recipients and curing large and metastatic tumors. Donor T cells were necessary for tumor protection, and CD8 T-cell immune responses were enhanced by posttransplantation booster vaccination. Hematopoietic stem cell transplantation is a standard therapy for lymphoma. Therefore, in situ tumor vaccination followed by immunotransplantation of harvested tumor-specific T cells could be directly tested in clinical trials to treat otherwise resistant malignancies.

Abstract

Polymorphisms of activating FcgammaRIIIa (CD16) and FcgammaRIIa (CD32a) have been found to predict rituximab response, probably because of the relative efficiency of different FcgammaR variants in performing antibody-dependent cellular cytotoxicity. The inhibitory FcgammaRIIb (CD32b) has an opposing effect on effector cells. Here, we examined whether an FcgammaRIIb 232 isoleucine (I)/threonine (T) polymorphism predicts rituximab response in 101 patients with follicular lymphoma. Eighty-four patients were 232 I/I, 15 were 232 I/T and two were 232 T/T. The response rate was similar among the three groups. The 2-year progression free survival (PFS) and median time to progression (TTP) were not different between I/I and I/T groups. The TTP was not determined in T/T group because of small number of patients. The FcgammaRIIIa 158 V/V and FcgammaRIIa 131 H/H genotypes continued to emerge as independent predictors for higher response rate and longer TTP. This study is the first to determine whether inhibitory FcgammaRIIb play a role in rituximab's anti-tumor effect in humans.

Abstract

There has been accumulating evidence that CD4(+)CD25(+) FoxP3 expressing regulatory T cells (Treg) are highly concentrated in tumors, thereby fostering an immune-privileged microenvironment. Some studies have shown that T-cell receptor (TCR) stimulation can convert conventional T cells into Treg. Follicular lymphoma (FL) B cells can enhance this Treg conversion. We investigated whether FL tumor B cells, as opposed to normal B cells, are unique in their ability to convert effector T cells into Treg. We found that tumor B cells alone, without artificial TCR stimulation, could induce conventional T cells to express FoxP3 and to acquire regulatory function. In contrast to their malignant counterpart, normal B cells did not induce Treg conversion. Treg conversion was independent of the T cell background, as T cells isolated from FL or normal peripheral blood were equally susceptible to being converted by tumor B cells. Our study provides evidence for a tumor-specific mechanism by which FL tumor cells promote immune escape through the induction of Treg.

Abstract

Recently, immunoglobulin G Fc receptor (FcgammaR) polymorphisms have been found to correlate with the clinical response to rituximab or idiotype vaccine in patients with follicular lymphoma. Two critical questions are whether the FcgammaR polymorphisms correlate with the clinical outcomes after chemotherapy alone in patients with follicular lymphoma and whether they can be explained by linking to underlying biology of follicular lymphoma. This is an important issue because the clinical decisions about the use of antibody therapy may be based on the FcgammaR polymorphisms of these patients. Here, we analyzed the FcgammaRIIIa 158 V/F, FcgammaRIIa 131 H/R, and FcgammaRIIb 232 I/T polymorphisms in a group of 188 patients with follicular lymphoma who were treated with chemotherapy without rituximab initially. In the current study, FcgammaR polymorphisms neither correlated with response rate or time to progression after induction chemotherapy, nor with time to the initial therapy or overall survival after diagnosis. Our results confirm that the correlation between FcgammaR polymorphisms and clinical outcome is specific to immunotherapy such as rituximab and idiotype vaccination, and not due to any effect on the underlying clinical behavior of the disease or chemotherapy response.

Abstract

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease characterized by variable clinical outcomes. Outcome prediction at the time of diagnosis is of paramount importance. Previously, we constructed a 6-gene model for outcome prediction of DLBCL patients treated with anthracycline-based chemotherapies. However, the standard therapy has evolved into rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Herein, we evaluated the predictive power of a paraffin-based 6-gene model in R-CHOP-treated DLBCL patients. RNA was successfully extracted from 132 formalin-fixed paraffin-embedded (FFPE) specimens. Expression of the 6 genes comprising the model was measured and the mortality predictor score was calculated for each patient. The mortality predictor score divided patients into low-risk (below median) and high-risk (above median) subgroups with significantly different overall survival (OS; P = .002) and progression-free survival (PFS; P = .038). The model also predicted OS and PFS when the mortality predictor score was considered as a continuous variable (P = .002 and .010, respectively) and was independent of the IPI for prediction of OS (P = .008). These findings demonstrate that the prognostic value of the 6-gene model remains significant in the era of R-CHOP treatment and that the model can be applied to routine FFPE tissue from initial diagnostic biopsies.

Abstract

The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab.DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome.In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis.We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

Abstract

Follicular lymphomas are frequently associated with the t(14;18)(q32;q21). This translocation can be detected by karyotype, polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). In addition to the breakpoints currently used for diagnosis located in the major breakpoint region (MBR) and the minor cluster region (mcr), recent studies have reported the existence of other breakpoints (3' BCL2, 5'mcr, and icr). In this study, we examined the frequency of all five breakpoints in 236 cases of follicular lymphomas by real-time PCR analysis. The distribution of breakpoint sites consisted of MBR in 118 cases (50%), mcr in 11 (5%), icr in 32 (13%), 3' BCL2 in 13 (6%), and 5' mcr in three cases (1%). These findings illustrate significantly higher frequency of the icr breakpoint as compared with the more frequently studied mcr. Correlation of breakpoints with histology showed that MBR breakpoints occur more frequently in grade 2 lymphomas (P = 0.042). A majority of the PCR-negative cases (75%) contained an IGH/BCL2 translocation with FISH methods, suggesting the presence of other BCL2 breakpoints. Correlation of breakpoints with survival did not reveal significant differences. Diagnostic laboratories should consider expanding their PCR methods to include other BCL2 breakpoints and correlating with FISH methods when appropriate.

Abstract

Established widely metastatic tumor was cured in a transplanted mouse B cell lymphoma model, by the combination of chemotherapy plus intratumoral injection of oligodeoxynucleotides containing unmethylated C-G motifs (CpG). This therapeutic effect required that the CpG be injected directly into the tumor and was dependent on CD8 T cells. Although the efficacy of CpG oligodeoxynucleotides has been thought to depend on the expression of TLR9, we unexpectedly found that tumor rejection did not require host expression of TLR9. By using a TLR9-deficient tumor and a TLR9KO host, we demonstrate that TLR9 expression either by the host or the tumor is required. These results indicate that activation of Ag presentation by cells within the tumor via TLR9 stimulation can be an effective form of immunotherapy. This study forms the basis of an ongoing clinical trial in patients with lymphoma.

Abstract

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 on antigen-presenting cells is essential for the initiation of cell-mediated and humoral immune responses. Malignant B cells also express CD40 and respond to CD40L by enhancing expression of costimulatory molecules. In this study, we investigated the therapeutic antitumor effect of intratumoral administration of recombinant fowlpox virus encoding murine CD40L (rF-mCD40L) in a murine B-cell lymphoma model. BALB/c mice with established s.c. and widely metastatic A20 lymphoma tumors were treated with intratumoral injections of rF-mCD40L together with systemic chemotherapy. This combined chemoimmunotherapy resulted in complete tumor regression and long-term survival of the mice. Some tumor cells in the injected sites expressed the CD40L transgene and had increased expression of the CD80 and CD86 costimulatory molecules. The therapeutic effect was dependent on CD8 but not on CD4 T cells. Moreover, there was a requirement that the recombinant CD40L virus be injected directly into the tumor, as opposed to peritumoral or distant sites. Thus, rF-mCD40L injected directly into the tumor microenvironment enhances the immunogenicity of tumor B cells. The results support future plans for intratumoral injection of rF-mCD40L in patients with lymphoma.

Abstract

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Abstract

We previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

Abstract

VICKZ family members are RNA-binding regulatory proteins expressed during embryogenesis but not usually found in normal adult tissue. The presence of VICKZ in normal germinal centers (GC) prompted us to characterize the expression pattern of this protein in lymphoid and hematopoietic tissues.We generated a pan-VICKZ antibody that recognized all three isoforms of VICKZ protein and screened 889 patients' samples by immunohistologic methods. We also analyzed the expression of VICKZ in normal hematopoiesis tissue by staining samples of tonsils, lymph nodesVICKZ protein expression was documented for the first time in normal human GC and in follicular (126/165), mediastinal large B-cell (9/10), Burkitt (2/2), diffuse large B-cell (DLBCL, 155/200), lymphocyte-predominant Hodgkin's (12/13), classical Hodgkin's (101/108), and anaplastic large cell (6/8) lymphomas and in lymphoid and myeloid leukemias. Since DLBCL may derive from GC or non-GC B cells we performed hierarchical cluster analysis for VICKZ, HGAL, BCL6, CD10, MUM1/IRF4 and BCL2 which showed that VICKZ is expressed in both subtypes. In addition, VICKZ mRNA isoforms were differentially expressed in lymphoma subtypes and over 40% of DLBCL expressed hVICKZ2, an isoform not usually present in normal GC B cells.We show that in normal lymphoid tissues VICKZ is expressed in GC lymphocytes but in lymphoid neoplasms its expression is not limited to GC-derived lymphoma subtypes. However, VICKZ exhibits differential expression in lymphoma subtypes and thus may be a marker of potential value in the diagnosis and study of hematopoietic neoplasia. The aberrant expression of its isoforms in DLBCL raises the possibility that these isoforms may be associated with different functions and suggests that further study of their role in normal and neoplastic lymphoid cells is warranted.

Abstract

We have reported that anti-idiotype antibody response and FcgammaRIIIa 158 valine/valine (V/V) genotype both correlate with better outcome in a group of 136 follicular lymphoma patients receiving idiotype vaccination after induction chemotherapy. Here, we examined whether this correlation is related in any way to the chemotherapy response. In patients with complete response (CR), the 5-year progression-free survival (PFS) was 69% for patients with antibody response and/or V/V genotype, while the PFS was only 40% for patients with neither; the median time to progression (TTP) was 10.47 versus 3.46 years (P=.012). In patients with partial response (PR), the 5-year PFS was 57% for patients with antibody response and/or V/V genotype, and 17% for patients with neither; the median TTP was not reached versus 1.31 years (P=.001). This study further confirms the strong association of clinical outcome with antibody response and with the functionally more active form of the Fc receptor in patients receiving idiotype vaccination regardless of their response to induction chemotherapy.

Abstract

The human germinal-center-associated lymphoma (HGAL) gene and its cognate protein are expressed in a germinal center (GC)-specific manner. Its expression in classic Hodgkin lymphoma (cHL) prompted us to address whether HGAL expression could distinguish biologically distinct subgroups of cHL. Tissue microarrays from 145 patients treated with curative intent showed HGAL staining in 75% and was closely correlated with MUM1/IRF4 (92%) expression. BCL6 (26%), CD10 (0%), BCL2 (31%), Blimp1 (0.02%), and Epstein-Barr virus (EBV) (20%) showed no specific correlation; neither did phospho-STAT6, a key mediator of IL-4 and IL-13 signaling that induces HGAL and is implicated in cHL pathogenesis. In our study cohort, the 5-year overall survival (OS) correlated with young age (less than 45 years, P < .001), low stage (stage I and II, P = .04), and low International Prognostic Score (P = .002). In univariate analysis, HGAL expression was associated with improved OS (P = .01) and failure-free survival (FFS) (P = .05) but was not independent of other factors in multivariate analysis of OS or FFS. The expression of the GC-specific marker HGAL in a subset of cHL suggests that these cHLs retain characteristics of GC-derived lymphomas. The association with improved OS in univariate but not multivariate analysis suggests that HGAL expression is related to known clinical parameters of improved survival.

Abstract

Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM, BCR cross-linking led to a quick burst of Syk, ERK1/2, and p38 signaling. In contrast, IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H(2)O(2) and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders, such as autoimmunity and cancer.

Abstract

Immunotherapy of cancer with DC vaccines has produced encouraging results in clinical trials. Antigen (Ag)-pulsed DC have elicited CD4+ and CD8+ T-cell immunity and tumor regression in humans. However, there is no standard method of DC production. The DC phenotype, number and Ag-loading process used in these studies have varied, making comparisons between trials difficult.In the present report a reproducible method was developed for the production of a DC-based vaccine. Monocytes were enriched by adhesion from healthy donor apheresis products and cultured with growth factors for maturation into DC. The cells were loaded with the tumor Ag idiotype proteins from patients with multiple myeloma. DC culture and Ag loading were performed in an automated and closed system. The DC product was characterized for phenotype by flow cytometry and for function in Ag uptake and Ag presentation.These monocyte-derived DC expressed high levels of costimulatory molecules (CD80/86). Ag-pulsed DC functioned to induce allogeneic proliferative lymphocyte responses and Ag-specific cytotoxic T lymphocyte (CTL) responses. The DC viability, phenotype and function were well preserved following prolonged frozen storage. Aliquots from the product of a single DC preparation could be used for sequential vaccinations without batch to batch variability.Ag-pulsed DC can be reproducibly generated for clinical use. These standardized methods are now being employed for a clinical trial to evaluate idiotype-pulsed DC vaccine therapy following non-myeloablative transplant for the treatment of multiple myeloma.

Abstract

Dendritic cells are potent antigen-presenting cells that can induce both immune responses and tolerance depending on their state of activation. Immunologic tolerance to established tumors is a major impediment for the development of effective cancer immunotherapy. Dendritic cells may be deficient in number or in function at the tumor site. To address this problem, we evaluated the ability of immature naïve dendritic cells to induce an antitumor immune response when injected directly into a murine B-cell lymphoma. Mice with advanced transplanted syngeneic tumor were given intratumoral injections of bone marrow-derived dendritic cells. Intratumoral dendritic cell injection alone had no antitumor effect. Systemic chemotherapy alone resulted in only transient tumor regression. However, the intratumoral injection of dendritic cells after chemotherapy led to complete, long-term tumor regression in the majority of treated mice. This dendritic cell-mediated antitumor effect was systemic, resulting in simultaneous elimination of the tumor at second uninjected sites. In addition, it resulted in long-term memory with resistance to tumor rechallenge. Both CD4+ and CD8+ T cells are necessary for the antitumor effect. Furthermore, tumors that occasionally recurred in mice with initial complete tumor regression could be retreated by the same combined chemoimmunotherapy approach. These results show that immunotherapy can succeed in the setting of advanced lymphoma if dendritic cells are restored and loaded with tumor antigens in situ at a single tumor site.

Abstract

We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

Abstract

The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.

Abstract

The unique immunoglobulin idiotype (Id) expressed by each B-cell lymphoma is a target for immunotherapy. Vaccination with Id induces humoral and/or cellular anti-Id immune responses. However, the clinical impact of these anti-Id immune responses is unknown. We and others have previously reported that immunoglobulin G Fc receptor (FcgammaR) polymorphisms predict the clinical response of lymphoma patients to passive anti-CD20 antibody infusions. In this study, we tested whether anti-Id immune responses or FcgammaR polymorphisms associate with clinical outcome of patients who received Id vaccination.We analyzed 136 patients with follicular lymphoma who had received Id vaccination. The anti-Id immune responses were measured and FcgammaRIIIa and FcgammaRIIa polymorphisms were determined and correlated with clinical outcome for these patients.Patients who mounted humoral immune responses had a longer progression-free survival (PFS) than those who did not (8.21 v 3.38 years; P = .018). Patients with FcgammaRIIIa 158 valine/valine (V/V) genotype also had a longer PFS than those with valine/phenylalanine (V/F) or phenylalanine/phenylalanine (F/F) genotypes (V/V, 8.21 v V/F, 3.38 years; P = .004; v F/F, 4.47 years; P = .035). Multivariate analysis using the Cox proportional hazards model showed that V/V genotype and humoral immune responses were independent positive predictors for PFS.This study is the first to identify the predictive value of FcgammaR polymorphism on clinical outcome in patients who received active immunotherapy with tumor antigen vaccines. Our results imply that the antibodies induced against a tumor antigen are beneficial and that FcgammaR-bearing cells mediate an antitumor effect by killing antibody-coated tumor cells.

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.

Abstract

Activation-induced cytidine deaminase (AID), highly expressed in germinal center (GC)-lymphocytes, is involved in somatic hypermutation (SHM). We examined AID expression in diffuse large B-cell lymphomas (DLBCL) of germinal center B-cell (GCB)-like and activated B-cell (ABC)-like subtypes. These two types of DLBCL are characterized by high and low expression of GC-specific genes, respectively. AID expression was detected in both GCB- and ABC-like DLBCL, thus demonstrating a dissociation between AID expression and that of other GC genes. We also tested for the presence of intraclonal heterogeneity in immunoglobulin and BCL6 genes in those same tumors and in follicle center lymphomas (FCL) that transformed to DLBCL. The level of AID expression did not correlate with the presence of intraclonal sequence heterogeneity in either IgV(H) or BCL6. Our findings suggest that lymphomas maintain some but not all of the gene expression signatures of their normal B-cell counterparts. The fact that AID expression can be elevated without intraclonal sequence heterogeneity raises the possibility that other factors are required for SHM in these tumors. We found decreased levels of AID expression in DLBCL that evolved from FCL and which had acquired new mutations in their BCL6 genes. This dissociation suggests that AID expression and SHM may occur at the time prior to the clinical detection of transformed lymphoma.

Abstract

Several gene-expression signatures can be used to predict the prognosis in diffuse large-B-cell lymphoma, but the lack of practical tests for a genome-scale analysis has restricted the use of this method.We studied 36 genes whose expression had been reported to predict survival in diffuse large-B-cell lymphoma. We measured the expression of each of these genes in independent samples of lymphoma from 66 patients by quantitative real-time polymerase-chain-reaction analyses and related the results to overall survival.In a univariate analysis, genes were ranked on the basis of their ability to predict survival. The genes that were the strongest predictors were LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2. We developed a multivariate model that was based on the expression of these six genes, and we validated the model in two independent microarray data sets. The model was independent of the International Prognostic Index and added to its predictive power.Measurement of the expression of six genes is sufficient to predict overall survival in diffuse large-B-cell lymphoma.

Abstract

The T cell receptor (TCR) idiotype on T cell lymphomas can serve as a vaccine target. To clone the relevant genes, 5' rapid amplification of cDNA ends (RACE) was performed on 13 T cell lymphomas and nine control samples. Two polymerase chain reactions (PCR) were performed for each TCR chain (alpha and beta) and the proportion of the clonal TCR sequence over the total number of TCR sequences was calculated. For alpha, the average proportions were 0.43 vs. 0.05. For beta these were 0.44 and 0.04. The TCR was identified in 10 of 13 lymphoma samples.

Abstract

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8alpha knockout, micro MT, and FcgammaR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1(-/-) splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-gamma knockout mice demonstrated a requirement for IFN-gamma, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-gamma knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.

Abstract

Although rituximab is now routinely used in the treatment of B-cell non-Hodgkin's lymphoma, the mechanism of its antitumor effect is not clear. One potential mechanism of action involves antibody-dependent cellular cytotoxicity (ADCC). Two aspects of ADCC influence the effectiveness of this process: the susceptibility of tumor cells and the activation of effector cells via their immunoglobulin G fragment C receptors (Fc gamma Rs). Several Fc gamma R polymorphisms have been identified that may affect the killing function of natural killer cells and macrophages.The pretreatment tumor cells from 43 patients with follicular lymphoma were tested for their intrinsic susceptibility to rituximab-mediated ADCC. In addition, the Fc gamma RIIIa (CD16) and Fc gamma RIIa (CD32) polymorphisms were determined in an expanded group of 87 patients. The results were then correlated with clinical outcome of these patients.No difference was found between the susceptibility of tumors from patients who clinically responded to rituximab versus those who did not respond. Conversely, both the Fc gamma RIIIa 158 valine/valine and the Fc gamma RIIa 131 histidine/histidine genotypes were found to be independently associated with the response rate and freedom from progression.These data support the hypothesis that ADCC plays an important role in the clinical effect of rituximab at the level of the effector cell. It will be important to include information on Fc receptor polymorphisms in future trials of rituximab therapy.

Abstract

Follicular lymphoma (FL) is characterized by a relatively indolent clinical course, but the disease often transforms into a more aggressive large cell lymphoma with a rapidly progressive clinical course. In the present study, we analyzed 41 cases of FL known to have subsequently transformed to aggressive lymphoma and an additional 64 FL samples from patients not subsequently transformed. We studied BCL6 gene rearrangement by the methodology of long-distance inverse polymerase chain reaction (LDI-PCR). Of the 41 cases known to transform, 16 (39.0%) harbored BCL6 translocation or deletion at the time of FL diagnosis. Among 64 cases not known to transform, BCL6 translocation was detected in 9 (14.1%). The prevalence of BCL6 translocation in the group known to transform was significantly higher (P =.0048). Among the transformation cases, the partners of the BCL6 translocation were identified in 13 cases and included IGH, CIITA, U50HG, MBNL, GRHPR, LRMP, EIF4A2, RhoH/TTF, and LOC92656 (similar to NAPA), whereas in the control group the BCL6 partners were IGH, CIITA, SIAT1, and MBNL. In 13 cases paired specimens before and after transformation were available. Among these paired specimens, a loss (3 cases) or a gain (1 case) of BCL6 translocation was observed after the transformation. Analysis of clonality showed that all of these cases represented the evolution of a subclone of the original tumor population. Our study demonstrated that BCL6 translocation is not necessary for transformation but that BCL6 translocation in FL may constitute a subgroup with a higher risk to transform into aggressive lymphoma.

Abstract

The FAS antigen (CD95/APO-1) is suggested to be a tumor suppressor gene since mice and patients with congenital FAS mutations are prone to B cell lymphomas and somatic FAS mutations are described in hematological and solid tumors. Indeed, mutations of the FAS antigen have been found in 13% of multiple myelomas, 6% of follicle center lymphomas (FCL) and 21% of diffuse large B-cell lymphomas (DLBCL). To assess the possible role of FAS mutations in higher-grade transformation of FCL, biopsy specimens from 16 FCL patients were analyzed by denaturing high performance liquid chromatography and direct sequencing. Overall, 17 biopsy specimens obtained at the time of FCL diagnosis (2 biopsy specimens from one patient), 4 sequential biopsies obtained at the time of FCL relapse and 14 sequential biopsies from the time of morphologic transformation to DLBCL were evaluated. Ten polymorphisms were detected, only 4 of which have been reported previously. Nine of the polymorphisms occurred in non-translated regions, while one silent mutation was located in exon 7. Neither loss of heterozygosity nor occurrence of new mutations was observed upon higher-grade transformation of FCL to DLBCL.

Abstract

The BCL6 gene, which functions as a transcription repressor, is the target of multiple chromosomal translocations in non-Hodgkin's lymphomas (NHL). These translocations occur in the nontranslated region of the BCL6 gene, juxtaposing regulatory sequences of the diverse partner genes to the open reading frame of the BCL6 gene and thus are thought to deregulate BCL6 gene expression. The levels of expression of the BCL6 gene and protein have been demonstrated to predict the clinical outcome of diffuse large B-cell lymphomas. By contrast, the prognostic significance of BCL6 gene translocations is unclear. In this study we have sought an explanation for this apparent discrepancy. We examined tumors with a variety of different BCL6 translocations and therefore with a variety of potentially substituted promoters. We found no increase in total BCL6 mRNA levels in the NHL specimens harboring BCL6 gene translocation. Indeed, some of these tumors expressed relatively low quantities of the BCL6 mRNA. We also sought to determine whether BCL6 transcription occurs from the rearranged or from the normal untranslocated allele in these tumors. We demonstrate that lymphoma cell lines and majority of NHL tumor specimens expressed BCL6 mRNA predominantly from the rearranged allele that may come under the control of various partner gene promoters. However, few NHL tumors with BCL6 gene translocations expressed BCL6 mRNA equally from the rearranged and the nonrearranged alleles. Neither the nature of the substituted promoters nor the presence of activating mutations in the BCL6 regulatory sequences correlated with the allelic expression of the BCL6 gene in these tumors.

Abstract

Higher grade histological transformation of follicular lymphoma (FL) to more aggressive diffuse large B-cell lymphomas (DLBCL) occurs in 10-60% of the cases. Review of the current knowledge of genetic and molecular alterations associated with the higher grade transformation of FCL suggests that the process that leads to clinically and phenotypically similar end-point can occur by functionally diverse genetic lesions. The most commonly identified genetic alterations associated with the FCL transformation are TP53 gene mutations, inactivation of CDKN2A and CDKN2B genes and deregulation of the C-MYC gene. These lesions affect different aspects of normal cell physiology (apoptosis, cell cycle control, and proliferation) and are potential targets for gene-specific therapies.

Abstract

Analysis of global gene expression with DNA microarrays has great potential to improve the understanding of tumorigenesis advance tumor diagnosis and classification, and affect cancer treatment. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. However, we now realize that the disease is extremely heterogeneous. This review summarizes the progress in understanding DLBCL that has been made as a result of the application of gene expression profiling.

Abstract

The costimulatory molecules B7-1, intercellular adhesion molecule-1 (ICAM-1), and leukocyte function-associated antigen-3 (LFA-3) play pivotal roles in the activation of T cells. We investigated whether in vivo vaccination with lymphoma cells infected with a recombinant, nonreplicating fowlpox (FP) virus encoding this triad of costimulatory molecules (TRICOM) could stimulate lymphoma-specific immunity.TRICOM-infected A20 B lymphoma cells were analyzed for expression of B7-1, ICAM-1, and LFA-3. Mice (10 per group) were vaccinated with irradiated A20 cells infected with either the TRICOM vector or the wild-type FP virus (WT-FP), challenged with live A20 tumor cells, and followed for survival. Mice with established A20 tumors were also treated with irradiated TRICOM-infected A20 cells. Survival curves were compared with the log-rank statistic. The mechanism of the antitumor effect was studied by in vivo depletion of CD4(+) and CD8(+) T cells and in vitro cytotoxicity assays. All statistical tests were two-sided.A20 tumor cells infected with TRICOM expressed high levels of B7-1, ICAM-1, and LFA-3. Mice vaccinated with irradiated TRICOM-infected A20 cells had prolonged survival relative to mice vaccinated with WT-FP-infected cells (80% versus 20% survival at 110 days; P

Abstract

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of non-Hodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, beta-actin, peptidylprolyl isomerase A, beta 2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, beta-glucoronidase and 18S ribosomal RNA). In all, 12 different B- and T-cell lymphoma/leukemia cell lines, 80 B- and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD(260) measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD(260) measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.

Variation in gene expression patterns in follicular lymphoma and the response to rituximabPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABohen, S. P., Troyanskaya, O. G., Alter, O., Warnke, R., Botstein, D., Brown, P. O., Levy, R.2003; 100 (4): 1926-1930

Abstract

Analysis of the patterns of gene expression in follicular lymphomas from 24 patients suggested that two groups of tumors might be distinguished. All patients, whose biopsies were obtained before any treatment, were treated with rituximab, a monoclonal antibody directed against the B cell antigen, CD20. Gene expression patterns in the tumors that subsequently failed to respond to rituximab appeared more similar to those of normal lymphoid tissues than to gene expression patterns of tumors from rituximab responders. These findings suggest the possibility that the response of follicular lymphoma to rituximab treatment may be predicted from the gene expression pattern of tumors.

Abstract

We have cloned and characterized a novel human gene, HGAL (human germinal center-associated lymphoma), which predicts outcome in patients with diffuse large B-cell lymphoma (DLBCL). The HGAL gene comprises 6 exons and encodes a cytoplasmic protein of 178 amino acids that contains an immunoreceptor tyrosine-based activation motif (ITAM). It is highly expressed in germinal center (GC) lymphocytes and GC-derived lymphomas and is homologous to the mouse GC-specific gene M17. Expression of the HGAL gene is specifically induced in B cells by interleukin-4 (IL-4). Patients with DLBCL expressing high levels of HGAL mRNA demonstrate significantly longer overall survival than do patients with low HGAL expression. This association was independent of the clinical international prognostic index. High HGAL mRNA expression should be used as a prognostic factor in DLBCL.

Abstract

B-cell lymphomas express tumor-specific immunoglobulin, the variable regions of which [idiotype (Id)] can serve as a target for active immunotherapy. Promising results have been obtained in clinical studies of Id vaccination using Id proteins.However, Id protein is laborious and time-consuming to produce. DNA vaccination is an attractive alternative for delivering Id vaccines, because Id DNA can be rapidly isolated by PCR techniques. DNA coding for lymphoma Id can provide protective immunity in murine models. In the present study, we performed a Phase I/II clinical trial to study the safety and immunogenicity of naked DNA Id vaccines in 12 patients with follicular B-cell lymphoma. The DNA encoded a chimeric immunoglobulin molecule containing variable heavy and light chain immunoglobulin sequences derived from each patient's tumor, linked to the IgG2a and kappa mouse immunoglobulin (MsIg) heavy- and light-chain constant regions chains, respectively. Patients in remission after chemotherapy received three monthly i.m. injections of the DNA in three dose escalation cohorts of four patients each (200, 600, and 1800 micro g). After vaccination, 7 of 12 patients mounted either humoral (n = 4) or T-cell-proliferative (n = 4) responses to the MsIg component of the vaccine. In one patient, a T-cell response specific to autologous Id was also measured. Anti-Id antibodies were not detectable in any patient. A second series of vaccinations was then administered using a needle-free injection device (Biojector) to deliver 1800 micro g both i.m. and intradermally (i.d.); 9 of 12 patients had humoral (n = 6) and/or T-cell (n = 4) responses to MsIg. Six of 12 patients exhibited humoral and/or T-cell anti-Id responses; yet, these were cross-reactive with Id proteins from other patient's tumors. Subsequently, a third series of vaccinations was carried out using 500 micro g of human granulocyte-macrophage colony-stimulating factor DNA mixed with 1800 micro g of Id DNA. The proportion of patients responding to MsIg remained essentially unchanged (8 of 12), although humoral or T-cell responses were boosted in some cases. Throughout the study, no significant side effects or toxicities were observed. Despite the modest level of antitumor immune responses in this study, DNA vaccine technology retains potential advantages in developing anti-Id immunotherapies. Additional studies are warranted to optimize vaccine dose, routes of administration, vector designs, and prime-boost strategies. These results will help guide the design of such future DNA vaccine trials.

Abstract

Follicle center lymphoma (FCL) is an indolent low-grade B cell non-Hodgkin's lymphoma (NHL) that frequently transforms to aggressive diffuse large B cell lymphoma (DLBCL). Histological transformation of FCL is commonly associated with accumulation of secondary genetic alterations. The BCL-6 gene is commonly implicated in the pathogenesis of DLBCL and its expression may be altered by clonal rearrangements and somatic point mutations in its 5' non-translated regulatory region. Recently, somatic mutations of the BCL-6 gene were associated with the transformation process. Here, we examined BCL-6 mRNA expression and BCL-6 mutations in paired biopsies from the same patients obtained at the time of FCL diagnosis and after transformation. BCL-6 mRNA expression markedly increased upon transformation (1.9- to 4.8-fold) in three cases, remained unchanged in one case and decreased compared to the diagnosis FCL specimens in four cases. The three specimens that demonstrated an increase in the BCL-6 mRNA expression upon transformation harbored BCL-6 gene mutations in the 5' region of the first intron that overlapped with the previously reported negative regulatory region of the gene. Accumulation of new mutations in this region was not observed in DLBCL biopsies in which the BCL-6 mRNA expression did not increase. The present study demonstrates that although BCL-6 gene mutations do accumulate during the transformation process and, depending on their location within the first intron, may deregulate BCL-6 mRNA expression, increase in BCL-6 mRNA expression is not uniformly required for transformation from FCL to DLBCL.

Abstract

The natural history of follicular lymphoma (FL) is frequently characterized by transformation to a more aggressive diffuse large B cell lymphoma (DLBCL). We compared the gene-expression profiles between transformed DLBCL and their antecedent FL. No genes were observed to increase or decrease their expression in all of the cases of histological transformation. However, two different gene-expression profiles associated with the transformation process were defined, one in which c-myc and genes regulated by c-myc showed increased expression and one in which these same genes showed decreased expression. Further, there was a striking difference in gene-expression profiles between transformed DLBCL and de novo DLBCL, because the gene-expression profile of transformed DLBCL was more similar to their antecedent FL than to de novo DLBCL. This study demonstrates that transformation from FL to DLBCL can occur by alternative pathways and that transformed DLBCL and de novo DLBCL have very different gene-expression profiles that may underlie the different clinical behaviors of these two types of morphologically similar lymphomas.

Abstract

CD40-CD40 ligand (CD40L) interactions play a critical role in the activationof cellular immunity. CD40L enhances the antigen presentation function of CD40-expressing B cells. We have used a murine B-cell lymphoma model (A20) to study the in vivo antitumor effect of the administration of tumor cells transduced with a recombinant adenovirus encoding CD40L (AdvCD40L). After infection with AdvCD40L, A20 tumor cells up-regulate several T-cell costimulatory molecules (CD80, CD86, ICAM-1, and LFA-3) and Fas expression. Animals vaccinated with irradiated tumor cells transduced with AdvCD40L are protected against a lethal dose of parental A20 tumor cells. Animals with pre-existing tumors treated with AdvCD40L-transduced tumor cells display inhibition of the tumor growth, and this treatment confers a survival advantage. In vivo depletion studies demonstrate that both CD4(+) and CD8(+) T cells mediate the antitumor immunity provided by AdvCD40L-transduced tumor cells. These results show that genetic modification of tumor B cells with CD40L can be a useful strategy to promote systemic immunity against B-cell malignancies and provide an in vivo system to allow for additional evaluation and refinement of this approach.

Abstract

Loss of function of the ataxia-telangiectasia mutated (ATM) gene, located on human chromosome 11q22-23, is the cause of ataxia-telangiectasia (A-T), which is associated with an extremely high risk for lymphoma. Abnormalities in 11q22-23, including deletions and mutations of the ATM gene, have been reported in T-cell prolymphocytic leukemias, B-CLL and in mantle cell lymphoma. In a survey of gene expression in follicle center lymphomas (FCL) and diffuse large B-cell lymphomas (DLBCL), almost all FCL expressed significant levels of ATM and the majority of DLBCL expressed low levels of ATM. This finding raised the possibility that the transformation of some FCL to DLBCL might be associated with inactivation of the ATM gene. Therefore, we analyzed biopsy specimens of 17 patients with FCL obtained at the time of diagnosis, four subsequent biopsies obtained at the time of FCL relapse and seven subsequent biopsies at the time of transformation to DLBCL. A comprehensive analysis of the ATM gene was performed by denaturing high performance liquid chromatography and sequencing. The analysis covered all of the 66 exons including the 9168 base pairs of ATM coding sequence as well as 16,676 base pairs of non-coding sequence. Twenty-eight known polymorphisms and rare sequence variants were observed, but no classic A-T mutations were detected. In 11 tumors, both tumor B-cells and normal T-cells were sorted for separate examination, and in each case, polymorphisms and rare variants were present in both tumor and normal cells. No new ATM gene mutations were associated with transformation from FCL to DLBCL. Thus, ATM gene mutations do not play a pivotal role either in the pathogenesis of FCL or in its transformation to DLBCL.

Abstract

Tumor-specific clonal immunoglobulin expressed by B-cell lymphomas (idiotype [Id]) can serve as a target for active immunotherapy. We have previously described the vaccination of 4 patients with follicular lymphoma using dendritic cells (DCs) pulsed with tumor-derived Id protein and now report on 35 patients treated using this approach. Among 10 initial patients with measurable lymphoma, 8 mounted T-cell proliferative anti-Id responses, and 4 had clinical responses--2 complete responses (CRs) (progression-free [PF] for 44 and 57 months after vaccination), 1 partial response (PR) (PF for 12 months), and 1 molecular response (PF for 75+ months). Subsequently, 25 additional patients were vaccinated after first chemotherapy, and 15 of 23 (65%) who completed the vaccination schedule mounted T-cell or humoral anti-Id responses. Induction of high-titer immunoglobulin G anti-Id antibodies required coupling of Id to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH). These antibodies could bind to and induce tyrosine phosphorylation in autologous tumor cells. Among 18 patients with residual tumor at the time of vaccination, 4 (22%) had tumor regression, and 16 of 23 patients (70%) remain without tumor progression at a median of 43 months after chemotherapy. Six patients with disease progression after primary DC vaccination received booster injections of Id-KLH protein, and tumor regression was observed in 3 of them (2 CRs and 1 PR). We conclude that Id-pulsed DC vaccination can induce T-cell and humoral anti-Id immune responses and durable tumor regression. Subsequent boosting with Id-KLH can lead to tumor regression despite apparent resistance to the primary DC vaccine.

Abstract

Regeneration of hematopoiesis after allogeneic hematopoietic cell transplantation (HCT) involves conversion of the recipient's immune system to donor type. It is likely that distinct cell lineages in the recipient reconstitute at different rates. Dendritic cells (DCs) are a subset of hematopoietic cells that function as a critical component of antigen-specific immune responses because they modulate T-cell activation, as well as induction of tolerance. Mature DCs are transferred with hematopoietic grafts and subsequently arise de novo. Little information exists about engraftment kinetics and turnover of this cell population in patients after allogeneic HCT. This study examined the kinetics of DC chimerism in patients who underwent matched sibling allogeneic HCT. T-cell, B-cell, and myelocytic and monocytic chimerism were also studied. Peripheral blood cells were analyzed at defined intervals after transplantation from 19 patients with various hematologic malignancies after treatment with myeloablative or nonmyeloablative preparatory regimens. Cell subsets were isolated before analysis of chimerism. Despite the heterogeneity of the patient population and preparatory regimens, all showed rapid and consistent development of DC chimerism. By day +14 after transplantation approximately 80% of DCs were of donor origin with steady increase to more than 95% by day +56. Earlier time points were examined in a subgroup of patients who had undergone nonmyeloablative conditioning and transplantation. These data suggest that a major proportion of blood DCs early after transplantation is donor-derived and that donor chimerism develops rapidly. This information has potential implications for manipulation of immune responses after allogeneic HCT.

Abstract

ASPP2 interacts with the tumor suppressor protein p53, promotes damage-induced apoptosis, and can specifically stimulate p53 apoptotic function. Thus, ASPP2 may function as a tumor suppressor and/or play a role in the cellular response to cytotoxic injury. To explore the role of ASPP2 in human cancer, we determined ASPP2 expression in two lymphoma subtypes with differing clinical outcomes: diffuse large B-cell lymphoma (DLBCL) and follicular center lymphoma (FCL). A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect ASPP2 mRNA. Sixty-one DLBCL and twenty-three FCL cases were analyzed and normalized ASPP2 levels were expressed relative to an mRNA standard. We found that ASPP2 mean expression strongly correlated with lymphoma subtype: DLBCL = 11.74 and FCL = 4.99 (p = 0.029, unpaired 2-tailed t-test). Importantly, ASPP2 expression was variable in DLBCL but not FCL (DLBCL-range, 0.04-94.6; FCL-range, 1.2-15.0). In these DLBCL cases, serum lactate dehydrogenase (LDH) was an independent predictor of survival with median survival in the high LDH group of 24 months and median survival not achieved in the normal-low LDH group (p = 0.014, Log-Rank Test). Mean ASPP2 levels trended toward an inverse correlation with LDH levels: High LDH, ASPP2 = 6.2; Normal-low LDH, ASPP2 = 18.2 (p = 0.074, unpaired 2-tailed t-test). In the DLBCL cases with ASPP2 levels > 7.8, only 10% (1/10) had a high LDH, in contrast to cases with ASPP2 levels < 7.8 in which 59% (26/44) had a high LDH (p = 0.011, Fisher Exact Test). Thus, low ASPP2 mRNA levels may correlate with poor clinical outcome in lymphoma which is consistent with the hypothesis that ASPP2 may play a role in tumor formation and/or sensitivity to cytotoxic agents. Larger studies as well as analysis of different tumor types are warranted.

Abstract

Follicle center lymphoma (FCL) accounts for approximately 40% of all non-Hodgkin's lymphomas (NHL). The genetic-environmental interactions involved in the etiology and pathogenesis of this disease are unknown. In our previous study a single nucleotide polymorphism (SNP) (397C) in the regulatory untranslated first intron region of the BCL-6 gene was found in four of the eight FCL patients but in none of the 10 healthy controls. To further evaluate the potential association between the 397C allele of the BCL-6 gene and FCL, we performed a case-control study. Genomic DNA was isolated from 85 FCL patients, from 98 control cases without a previous history of malignancy, treated at Stanford University Medical Center for non-malignant disorders and from 90 samples from the DNA Polymorphism Discovery Resource. The 397G and the 397C polymorphic alleles were identified by a PCR-RFLP method. To evaluate the possible effect of this polymorphism on gene expression, BCL-6 mRNA levels in nine FCL tumors with the 397G-G genotype and in nine FCL tumors with the 397G-C genotype were measured by quantitative real-time RT-PCR. The 397C polymorphic allele was found in 32 FCL cases (37.6%), in 20 controls (20.4%) and in 17 (18.9%) samples from the DNA Polymorphism Discovery Resource. The prevalence of the 397G-C and 397C-C genotypes was significantly higher in FCL cases than in control group (p = 0.01). No difference in BCL-6 gene expression was observed between FCL cases with 397G-G and 397G-C genotypes. The present study demonstrates a possible association between the 397C allele of the BCL-6 proto-oncogene and FCL. The similar levels of BCL-6 mRNA expression in 397G-G and in 397G-C FCL cases suggests that any possible oncogenic effect of the polymorphic allele would not simply be related to a direct effect on BCL-6 gene expression and suggests the existence of other FCL susceptibility genes that are in linkage disequilibrium with the 397C allele of the BCL-6 gene.

Abstract

Rituximab is a chimeric monoclonal antibody that targets B-cell-specific antigen CD20 and an effective treatment for B-cell non-Hodgkin lymphoma. Although it is readily used in clinical practice, the exact mechanism of its antitumor effect is unclear. One potential mechanism involves complement-mediated cytotoxicity. It has been shown that rituximab induces complement-mediated cytotoxicity in follicular lymphoma cells in vitro, and complement inhibitors CD55 and CD59 may regulate this process. To determine whether complement inhibitors play a role in regulating the antitumor effect of rituximab, the expression of complement inhibitors CD46, CD55, and CD59 was analyzed in pretreatment tumor cells from 29 rituximab-treated follicular lymphoma patients. Among them, 8 patients achieved complete responses, 11 patients achieved partial responses, and 10 patients showed no or minimal responses to rituximab treatment. Expression of surface CD20, CD46, CD55, and CD59 was determined by 2-color flow cytometry. Although the CD59 level was slightly lower in the complete response group, there was no statistically significant difference in the expression of individual complement inhibitor CD46 (mean channel fluorescence [MCF]: NR, 26.4; PR, 21.9; CR, 29.9), CD55 (MCF: NR, 16.4; PR, 14.9; CR, 23.2), or CD59 (MCF: NR, 41.6; PR, 40.6; CR, 30.6), the combination of any 2 inhibitors, or all 3 on tumor cells from 3 response groups. In addition, there was no difference in the rituximab-induced complement-mediated cytotoxicity in an in vitro assay using tumor cells from 3 response groups. Thus, CD46, CD55, and CD59 expression on pretreatment tumor cells, or their susceptibility to in vitro complement-mediated killing, does not predict clinical outcome after rituximab treatment.

Abstract

Diffuse large B-cell lymphoma (DLBCL) is characterized by a marked degree of morphologic and clinical heterogeneity. Establishment of parameters that can predict outcome could help to identify patients who may benefit from risk-adjusted therapies. BCL-6 is a proto-oncogene commonly implicated in DLBCL pathogenesis. A real-time reverse transcription-polymerase chain reaction assay was established for accurate and reproducible determination of BCL-6 mRNA expression. The method was applied to evaluate the prognostic significance of BCL-6 expression in DLBCL. BCL-6 mRNA expression was assessed in tumor specimens obtained at the time of diagnosis from 22 patients with primary DLBCL. All patients were subsequently treated with anthracycline-based chemotherapy regimens. These patients could be divided into 2 DLBCL subgroups, one with high BCL-6 gene expression whose median overall survival (OS) time was 171 months and the other with low BCL-6 gene expression whose median OS was 24 months (P =.007). BCL-6 gene expression also predicted OS in an independent validation set of 39 patients with primary DLBCL (P =.01). BCL-6 protein expression, assessed by immunohistochemistry, also predicted longer OS in patients with DLBCL. BCL-6 gene expression was an independent survival predicting factor in multivariate analysis together with the elements of the International Prognostic Index (IPI) (P =.038). By contrast, the aggregate IPI score did not add further prognostic information to the patients' stratification by BCL-6 gene expression. High BCL-6 mRNA expression should be considered a new favorable prognostic factor in DLBCL and should be used in the stratification and the design of risk-adjusted therapies for patients with DLBCL. (Blood. 2001;98:945-951)

Abstract

Vaccination with tumor-specific immunoglobulin or idiotype (Id) is a promising new form of immunotherapy for B-cell malignancies. Id protein vaccination has demonstrated clinical activity in B-cell lymphomas, yet it requires the laborious and time-consuming procedures of tumor-myeloma cell hybridization, large-scale in vitro culture, and protein purification. Recombinant adenoviruses are highly efficient and immunogenic gene transfer vehicles from which individualized vaccines can be rapidly assembled using polymerase chain reaction-amplified tumor Id genes. Id-encoding adenoviruses were evaluated as vaccines in 2 murine B-cell lymphoma models. A single injection of recombinant Id adenovirus provided protection from subsequent tumor challenge that was equivalent or superior to that afforded by Id protein vaccination. Protected mice had substantial serum titers of Id-specific antibodies. When used in conjunction with chemotherapy, vaccination also prolonged the survival of mice bearing pre-existing tumor. Mechanistic studies demonstrated that tumor protection was not dependent upon T cells. Importantly, in mice prevaccinated with an irrelevant adenovirus, tumor protection following vaccination with Id adenovirus was not significantly impaired. These findings have implications for the design of future lymphoma immunotherapy trials.

Abstract

T cell lymphoproliferative disorders continue to be serious management problems, and so alternative therapeutic modalities are continuously being explored. One such strategy involves immunotherapy using the T cell receptor (TCR) as a target. Specifically we are attempting to develop a T cell receptor idiotype (TCR-Id) vaccine because the TCR-Id can serve as a tumor-specific antigen. In this article we will briefly review the rationale for TCR-Id vaccines, the preclinical models as developed in our laboratory, and a discussion of our current plans for a vaccine trial in mycosis fungoides.

Abstract

Vaccination with the idiotype (Id) protein derived from B-cell malignancies can produce Id-specific immune responses that correlate with improved remission duration and survival rates in patients with follicular non-Hodgkin's lymphoma (NHL). A state of minimal or no residual disease correlates strongly with the laboratory detection of a cellular or humoral immune response. High-dose cytotoxic therapy (HDCT) with autologous stem cell support (autologous bone marrow transplantation [ABMT]) can provide profound cytoreduction of B-cell NHL, but the potential immune suppression associated with myeloablative therapy may compromise a patient's ability to mount a specific immune response. To determine whether patients with NHL could mount detectable immuneresponses following ABMT, Id vaccines were administered at 2 to 12 months following myeloablative therapy to a series of patients with relapsed or resistant B-cell NHL. Two different vaccination strategies produced robust immune responses against KLH in all patients, supporting the capacity of the reconstituted immune system following HDCT to react against a strong antigen. Combining the results from both vaccination strategies, 10 of 12 patients mounted Id-specific humoral or cellular responses. Vaccinations were consistently well tolerated. Of the 12 patients, 7 have experienced prolonged remissions with a follow-up from HDCT ranging from 3 to more than 11 years. Our experience serves to document the ability of the recovering immune system to react against both self and xenotypic antigens and supports the feasibility and safety of antigen-specific vaccination following myeloablative therapy in patients with B-cell NHL.

Abstract

Analysis of somatic mutations in V regions of Ig genes is important for understanding various biological processes. It is customary to estimate Ag selection on Ig genes by assessment of replacement (R) as opposed to silent (S) mutations in the complementary-determining regions and S as opposed to R mutations in the framework regions. In the past such an evaluation was performed using a binomial distribution model equation, which is inappropriate for Ig genes in which mutations have four different distribution possibilities (R and S mutations in the complementary-determining region and/or framework regions of the gene). In the present work, we propose a multinomial distribution model for assessment of Ag selection. Side-by-side application of multinomial and binomial models on 86 previously established Ig sequences disclosed 8 discrepancies, leading to opposite statistical conclusions about Ag selection. We suggest the use of the multinomial model for all future analysis of Ag selection.

A perspective on monoclonal antibody therapy: Where we have been and where we are goingLevy, R.W B SAUNDERS CO-ELSEVIER INC.2000: 43-46

Abstract

The history of monoclonal antibody therapy for cancer includes many promising beginnings, a vast number of detours, and now a major success--perhaps soon to be joined by several more. Years of research by hundreds of investigators worldwide have led to a growing understanding of the principles that underlie successful monoclonal antibody therapy and that can be used to create new antibody-based agents. However, there are many remaining uncertainties about this mode of therapy, even for those agents that have already joined our therapeutic armamentarium. Several key questions will be identified and used to suggest avenues for future research.

The history of the development of vaccines for the treatment of lymphoma.Clinical lymphomaTimmerman, J. M., Levy, R.2000; 1 (2): 129-139

Abstract

Exploitation of the immune system is an attractive strategy for developing selective lymphoma therapies. In the past several decades, increased knowledge of tumor immunology has granted investigators the tools to formulate a variety of lymphoma-specific vaccines. Vaccines targeting the tumor-specific immunoglobulin (idiotype) of B-cell lymphomas were the first to be developed, owing to successful active vaccination studies in animal models and clinical studies of passive anti-idiotype monoclonal antibodies. In initial clinical trials, patient-specific idiotype vaccines have been found to induce anti-idiotype immune responses that correlate with improved disease-free and overall survival and the reduction of the level of detectable residual disease. More recent strategies for improving the potency and practicality of idiotype vaccines are utilization of dendritic cells, recombinant idiotype proteins, and DNA vaccination. Custom-made vaccines utilizing whole autologous tumor cells are also being developed. Given the exciting results of these early lymphoma vaccine studies and the accelerated pace of immunologic research, it is hoped that vaccines will someday expand the armamentarium of effective lymphoma therapies.

Abstract

This phase II trial investigated the safety and efficacy of re-treatment with rituximab, a chimeric anti-CD20 monoclonal antibody, in patients with low-grade or follicular non-Hodgkin's lymphoma who relapsed after a response to rituximab therapy.Fifty-eight patients were enrolled onto this study, and two were re-treated within the study. Patients received an intravenous infusion of 375 mg/m(2) of rituximab weekly for 4 weeks. All patients had at least two prior therapies and had received at least one prior course of rituximab, with a median interval of 14.5 months between rituximab courses.Most adverse experiences (AEs) were transient grade 1 or 2 events occurring during the treatment period. Clinically significant myelosuppression was not observed; hematologic toxicity was generally mild and reversible. No patient developed human antichimeric antibodies after treatment. The type, frequency, and severity of AEs in this study were not apparently different from those reported in the phase III trial of rituximab. The overall response rate in 57 assessable patients was 40% (11% complete response and 30% partial responses). Median time to progression (TTP) in responders and median duration of response (DR) have not been reached, but Kaplan-Meier estimated medians are 17.8 months (range, 5.4+ to 26.6 months) and 16.3 months (range, 3.7+ to 25.1 months), respectively. These estimated medians are longer than the medians achieved in the patients' prior course of rituximab (TTP and DR of 12.4 and 9.8 months, respectively, P: >.1) and in a previously reported phase III trial (TTP in responders and DR of 13.2 and 11.6 months, respectively). Responses are ongoing in seven of 23 responders.In this re-treatment population, safety and efficacy were not apparently different from those after initial rituximab exposure.

Abstract

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (V(H)) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Abstract

Follicle center lymphoma (FCL) is an indolent low-grade B-cell non-Hodgkin's lymphoma (NHL) that frequently transforms to aggressive diffuse large B-cell lymphoma (DLBCL). Histologic transformation of FCL is commonly associated with accumulation of secondary genetic alterations. The BCL-6 gene is altered by chromosomal rearrangements and mutations clustering in its 5' noncoding regulatory region in up to 70% of primary DLBCL, but in a significantly smaller subset of FCL. Previous studies have shown that both chromosomal rearrangements and mutations could deregulate BCL-6 expression. To evaluate the association between progressive accumulation of BCL-6 regulatory region mutations and the histologic transformation of FCL, we analyzed by extensive cloning and sequencing paired biopsy specimens obtained at the time of FCL diagnosis and transformation (6 patients) or FCL relapse (3 patients). In an additional patient, biopsy specimens obtained at the time of diagnosis, FCL relapse, and subsequent transformation to DLBCL were evaluated. The presence of identical mutations in the paired diagnosis and posttransformation DLBCL specimens confirmed the common clonal origin of both the pretransformation and the posttransformation lymphomas. No new mutations in the 5' noncoding regulatory region of the BCL-6 gene were detected in any of the specimens evaluated at the time of FCL relapse. In contrast, 5 of the 7 transformed specimens contained new mutations not found in the paired original biopsy specimens obtained at the time of FCL diagnosis or relapse. The number of these new mutations ranged from 1 to 6 per specimen. Some of the new mutations tended to cluster in certain areas of the 5' noncoding regulatory region of the BCL-6 gene. Our results show that transformation of FCL to DLBCL is associated with accumulation of new mutations in the 5' noncoding regulatory region of the BCL-6 gene, that by deregulation of the BCL-6 gene expression may play a role in lymphoma transformation. (Blood. 2000;96:635-639)

Abstract

Rituximab and IFN have each demonstrated single-agent activity in patients with low-grade non-Hodgkin's lymphoma (NHL). A single-arm, multicenter, Phase II trial was conducted to assess the safety and efficacy of combination therapy with rituximab and IFN-alpha-2a in 38 patients with relapsed or refractory, low-grade or follicular, B-cell NHL. IFN-alpha-2a [2.5 or 5 million units (MIU)] was administered s.c., three times weekly for 12 weeks. Starting on the fifth week of treatment, rituximab was administered by i.v. infusion (375 mg/m2) weekly for 4 doses. All 38 patients received four complete infusions of rituximab and were evaluable for efficacy, although 11 patients (29%) did not-receive all 36 injections of IFN. The mean number of IFN-alpha-2a injections was 31 doses; the mean total units received were 141 MIU (maximum, 180 MIU). The study treatment was reasonably well tolerated with no unexpected toxicities stemming from the combination therapy. No grade 4 events were reported. Frequent adverse events during the treatment period included asthenia (35 of 38 patients), chills (31 of 38), fever (30 of 38), headache (28 of 38), nausea (23 of 38), and myalgia (22 of 38). The overall response rate was 45% (17 of 38 patients); 11% had a complete response, and 34% had a partial response. The Kaplan-Meier estimates for the median response duration and the median time to progression in responders are 22.3 and 25.2 months, respectively. Further follow-up is needed to determine whether this treatment combination leads to a significantly longer time to progression than single-agent treatment with rituximab.

Abstract

Vaccination using recombinant tumor-derived T-cell antigen receptor (TCR) protein induces a protective, idiotype-specific immune response against a murine T-cell tumor. However, the technically demanding task of producing patient-specific, recombinant TCR protein restricts the translation of TCR vaccines for clinical use. We report here the development of an effective recombinant TCR adenovirus vaccine. Individual adenoviruses were constructed to encode a chimeric TCR derived from either tumor Valpha or Vbeta regions fused to xenogeneic human constant regions. Coinjection of the chimeric alpha- and the beta-TCR adenoviruses protected mice against tumors. The level of protection was comparable to that achieved by an optimized regimen of recombinant TCR protein vaccines. Tumor immunity induced by TCR adenoviruses required the xenogeneic constant regions and was mediated by CD8+ T cells. Independent vaccines consisting of adenovirus expressing either chimeric alpha- or beta-TCR chain also stimulated a protective immune response. Immunization with TCR adenovirus may offer a new efficacious, protein-free vaccination approach for the treatment of T-cell malignancies.

Abstract

The unique Ag-presenting capabilities of dendritic cells (DCs) make them attractive vehicles for the delivery of therapeutic cancer vaccines. While tumor Ag-pulsed DC vaccination has shown promising results in a variety of murine tumor models and early clinical trials, the optimal form of tumor Ag for use in DC pulsing has not been determined. We have studied DC vaccination using alternative forms of a soluble protein tumor Ag, the tumor-specific Ig idiotype (Id) expressed by a murine B cell lymphoma. Vaccination of mice with Id-pulsed DCs was able to induce anti-Id Abs only when the Id was modified to constitute a hapten-carrier system. DCs pulsed with Id proteins modified to include foreign constant regions, foreign constant regions plus GM-CSF, or linkage to keyhole limpet hemocyanin (KLH) carrier protein were increasingly potent in their ability to elicit anti-Id Abs. Vaccination with Id-KLH-pulsed DCs induced tumor-protective immunity superior to that obtained with Id-KLH plus a chemical adjuvant, and protection was not dependent upon effector T cells. Rather, protection was associated with the induction of high titers of anti-Id Abs of the IgG2a subclass, characteristic of a Th1 response. These findings have implications for the design of therapeutic Ag-pulsed DC vaccines for cancer immunotherapy in humans.

Abstract

Diffuse large B-cell lymphoma (DLBCL) is a common type of non-Hodgkin's lymphoma (NHL) that is highly heterogeneous from both clinical and histopathologic viewpoints. The immunoglobulin (Ig) heavy (H) chain variable region genes were examined in 71 patients with untreated primary DLBCL. Fifty-eight potentially functional V(H) genes were detected in 53 DLBCL cases; V(H) genes were nonfunctional in 9 cases and were not detected in an additional 9 cases. The use of V(H) gene families by DLBCL tumors was unbiased without overrepresentation of any particular V(H) gene or gene family. Analysis of Ig mutations in comparison to the most closely related germline gene disclosed mutated V(H) genes in all but 1 DLBCL case. More than 2% difference from the most similar germline sequence was detected in 52 potentially functional and the 8 nonfunctional V(H) gene sequences, whereas less than 2% difference from the germline sequence was observed in 3 V(H) gene isolates. Only 3 V(H) gene isolates were unmutated. No correlation was found between V(H) gene use, mutation level, and International Prognostic Index (IPI) or survival. Six of 8 tested tumors showed evidence of ongoing somatic mutations. Evidence for positive or negative antigen selection pressure was observed in 65% of mutated DLBCL cases. Our findings indicate that the etiology and the driving forces for clonal expansion are heterogeneous, which may explain the well-known clinical and pathologic heterogeneity of DLBCL. (Blood. 2000;95:1797-1803)

Abstract

The BCL-6 proto-oncogene is involved in the genesis of non-Hodgkin lymphoma (NHL). Rearrangements due to chromosomal translocations and somatic mutations of the 5' noncoding regulatory region of the BCL-6 gene are potential mechanisms for altering its expression in NHL. To further elucidate the nature of the somatic mutations in the regulatory region of this gene, we have studied 10 healthy donors and 11 NHL biopsy samples by extensive molecular cloning and sequencing. In addition, we analyzed the BCL-6 genes of tumor and nontumor cells from 2 of the cases. The germ line sequence of this region was defined, which differs in 7 positions from that previously reported. In addition, 1 polymorphic variation at position 397(G or C) was identified. Deletions, insertions, and repeated substitution mutations were detected among the molecular isolates in 8 tumor specimens, with a mutational incidence ranging from 1.3 x 10(-3) to 1.3 x 10(-2)/bp (base pair). A total of 20 distinct substitution mutations, 1 insertion and 3 deletions were observed. One of these deletion mutations and 2 of the substitutions were observed in more than 1 tumor specimen from different individuals. In 3 tumor samples, identical mutations affecting both alleles were observed. These findings suggest the presence of mutational hot spots and hot specific events, a finding supported by our compilation of previously published data. In 6 samples, the nucleotide sequences showed evidence of intraclonal heterogeneity, consistent with a stepwise ongoing mutational process affecting the BCL-6 gene in the tumor cells. These mutations accumulating in the regulatory region of the BCL-6 gene could play a role in lymphoma progression and in the transformation of follicular lymphomas to more aggressive large cell lymphomas. (Blood. 2000;95:1400-1405)

Abstract

Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.

Abstract

The idiotype (Id) determinants on the multiple myeloma immunoglobulin can serve as tumor-specific antigens. An anti-Id immune response may stem the growth of the malignant clone. We report on 26 patients treated at our institution with high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) and vaccinated with the Id protein. The patients received chemotherapy and PBPCT to establish a minimal residual disease state. After high-dose therapy, the patients received a series of monthly immunizations consisting of 2 intravenous infusions of dendritic cells (DCs) pulsed with either Id protein or Id coupled with keyhole limpet hemocyanin (KLH) as an immunogenic carrier protein, followed by subcutaneous boosts of Id-KLH conjugates. DCs were obtained in all patients from a leukapheresis product 3 to 9 months after PBPCT. Patients were observed for toxicity, immune responses, and tumor status. The DC infusions and the administration of Id-KLH boosts were well tolerated, with patients experiencing only minor and transient side effects. Of the patients, 24 of 26 generated a KLH-specific cellular proliferative immune response. Only 4 patients developed an Id-specific proliferative immune response. Three of these immune responders were in complete remission at the time of vaccination. A total of 17 patients are alive at a median follow-up of 30 months after transplantation. Id vaccination with autologous DCs is feasible for myeloma patients after transplantation. Id-specific cellular responses can be induced in patients who are in complete remission. Further studies are needed to increase the rate of anti-Id immune responses in patients who do not achieve complete remission.

Abstract

Various studies have used DNA vaccination as a method of immunizing against tumors (1-12). As with any tumor vaccine, one challenge is to find a truly tumor-specific antigen (13,14). The majority of immunologically targeted tumor antigens are also expressed on a subset of normal host cells. Examples of such antigens include prostate-specific antigen, and CD20, a B cell marker. Some tumor antigens are specific for activated cells of certain types, such as carcinoembryonic antigen (CEA) or the IL-2 receptor. These are often found on embryonic or fetal cells as well as tumor cells. The carbohydrate antigens of melanomas and the immunoglobulin (Ig) idiotype of B cell lymphomas represent tumor-specific antigens (TSA). Unfortunately, TSA have not been identified in more common malignancies. Furthermore, the antigenic determinants of known TSA may differ between patients; for example, the tumor idiotype (Id) of B cell lymphoma is highly patient-specific and must be determined for each case.

Abstract

Large gene expression studies, such as those conducted using DNA arrays, often provide millions of different pieces of data. To address the problem of analyzing such data, we describe a statistical method, which we have called 'gene shaving'. The method identifies subsets of genes with coherent expression patterns and large variation across conditions. Gene shaving differs from hierarchical clustering and other widely used methods for analyzing gene expression studies in that genes may belong to more than one cluster, and the clustering may be supervised by an outcome measure. The technique can be 'unsupervised', that is, the genes and samples are treated as unlabeled, or partially or fully supervised by using known properties of the genes or samples to assist in finding meaningful groupings.We illustrate the use of the gene shaving method to analyze gene expression measurements made on samples from patients with diffuse large B-cell lymphoma. The method identifies a small cluster of genes whose expression is highly predictive of survival.The gene shaving method is a potentially useful tool for exploration of gene expression data and identification of interesting clusters of genes worth further investigation.

Abstract

A phase II trial was performed to evaluate the safety and efficacy of rituximab, a chimeric anti-CD20 monoclonal antibody, in patients with bulky (> 10-cm lesion) relapsed or refractory low-grade or follicular non-Hodgkin's lymphoma (NHL).Thirty-one patients received intravenous infusions of rituximab 375 mg/m(2) weekly for four doses. All patients had at least one prior therapy (median, three; range, one to 13) and had progressive disease at study entry. Patients were a median of 4 years from diagnosis.No patient had treatment discontinued because of an adverse event. No patient developed human antichimeric antibody. The overall response rate in 28 assessable patients was 43% with a median time to progression of 8.1 months (range, 4.5 to 18.6+ months) and median duration of response of 5.9 months (range, 2.8 to 12.1+ months). The average decrease in lesion size in patients who achieved a partial response was 76%, and patients with stable disease had a decrease in average lesion size of 26%. Median serum antibody concentration was higher in responders compared with nonresponders, and a negative correlation was shown between antibody concentration and tumor bulk at baseline.Rituximab single-agent outpatient therapy is safe and shows significant clinical activity in patients with bulky relapsed or refractory low-grade or follicular B-cell NHL.

Abstract

Several studies have shown that immunization with DNA, which encodes the idiotypic determinants of a B cell lymphoma, generates tumor-specific immunity. Although induction of antiidiotypic Abs has correlated with tumor protection, the effector mechanisms that contribute to tumor protection have not been clearly identified. This study evaluated the tumor protective effects of humoral and cellular immune mechanisms recruited by idiotype-directed DNA vaccines in the 38C13 murine B cell lymphoma model. Antiidiotypic Abs induced by DNA vaccination supported in vitro complement-mediated cytotoxicity of tumor cells, and simultaneous transfer of tumor cells and hyperimmune sera protected naive animals against tumor growth. However, in vitro stimulation of immune splenocytes with tumor cells failed to induce idiotype-specific cytotoxicity, and following vaccination, depletion of CD4 or CD8 T cell subsets did not compromise protection. Furthermore, protection of naive recipients against tumor challenge could not be demonstrated either by a Winn assay approach or by adoptive transfer of spleen and lymph node cells. Thus, in this experimental model, current evidence suggests that the tumor-protective effects of DNA vaccination can be largely attributed to idiotype-specific humoral immunity.

Abstract

The idiotype (Id) determinant on the multiple myeloma (MM) protein can be regarded as a tumor-specific marker. Immunotherapy directed at the MM Id may stem the progression of this disease. We report here on the first 12 MM patients treated at our institution with high-dose therapy and peripheral blood stem cell transplantation (PBSCT) followed by Id immunizations. MM patients received PBSCT to eradicate the majority of the disease. PBSCT produced a complete response in 2 patients, a partial response in 9 patients and stable disease in 1 patient. Three to 7 months after high-dose therapy, patients received a series of monthly immunizations that consisted of two intravenous infusions of Id-pulsed autologous dendritic cells (DC) followed by five subcutaneous boosts of Id/keyhole limpet hemocyanin (KLH) administered with adjuvant. Between 1 and 11 x 10(6) DC were obtained by leukapheresis in all patients even after PBSCT. The administration of Id-pulsed DC and Id/KLH vaccines were well tolerated with patients experiencing only minor and transient side effects. Two of 12 patients developed an Id-specific, cellular proliferative immune response and one of three patients studied developed a transient but Id-specific cytotoxic T-cell (CTL) response. Eleven of the 12 patients generated strong KLH-specific cellular proliferative immune responses showing the patients' immunocompetence at the time of vaccination. The two patients who developed a cellular Id-specific immune response remain in complete remission. Of the 12 treated patients, 9 are currently alive after autologous transplantation with a minimum follow-up of 16 months, 2 patients died because of recurrent MM and 1 patient succumbed to acute leukemia. These studies show that patients make strong anti-KLH responses despite recent high-dose therapy and that DC-based Id vaccination is feasible after PBSCT and can induce Id-specific T-cell responses. Further vaccine development is necessary to increase the proportion of patients that make Id-specific immune responses. The clinical benefits of Id vaccination in MM remain to be determined.

Abstract

Human tumors express a number of protein antigens that can be recognized by T cells, thus providing potential targets for cancer immunotherapy. Dendritic cells (DCs) are rare leukocytes that are uniquely potent in their ability to present antigens to T cells, and this property has prompted their recent application to therapeutic cancer vaccines. Isolated DCs loaded with tumor antigen ex vivo and administered as a cellular vaccine have been found to induce protective and therapeutic anti-tumor immunity in experimental animals. In pilot clinical trials of DC vaccination for patients with non-Hodgkin's lymphoma and melanoma, induction of anti-tumor immune responses and tumor regressions have been observed. Additional trials of DC vaccination for a variety of human cancers are under way, and methods for targeting tumor antigens to DCs in vivo are also being explored. Exploitation of the antigen-presenting properties of DCs thus offers promise for the development of effective cancer immunotherapies.

Abstract

The immunoglobulin on the surface of B-cell lymphomas can be a tumor-specific target for monoclonal antibody therapy. Between 1981 and 1993, 45 individuals with low grade B-cell lymphoma were treated with 52 courses of custom-made anti-idiotype antibodies. The antibodies were used either alone or in combination with alpha-interferon, chlorambucil, or interleukin-2 (IL-2). The majority of these patients responded to treatment, with a 66% overall and 18% complete response rate. Six patients (13%) experienced prolonged complete remissions, five of which are ongoing from 4 to 10 years after therapy and are the subject of this report. We asked whether residual lymphoma could be found in these patients with prolonged remissions. We performed enzyme-linked immunosorbent assay (ELISA) assays for idiotype protein or anti-idiotype antibodies in serum. Blood and bone marrow samples were examined by flow cytometry for idiotype positive cells, and by polymerase chain reaction (PCR) for clonal gene rearrangements of immunoglobulin CDR3 sequences or t(14;18) translocations. Using these sensitive and specific tests it was possible to detect very low levels of residual lymphoma in five of these patients who had been in clinical remission for 3 to 8 years before this evaluation. These five have continued without recurrence for up to 3 years since. Thus, we have found a pattern of residual inactive disease in patients treated with anti-idiotype antibodies. The biology of follicular lymphoma evidently includes the potential for tumor dormancy after therapies with varied mechanisms of action, resulting in clinical inactivity for many years. Thus, long-term control of the disease is possible at a clinical level despite persistence of the malignant clone.

Abstract

Radiosurgery is generally effective in obliterating true arteriovenous malformations, but less is known about its effects on angiographically occult vascular malformations (AOVMs). Since July 1983, 57 patients with surgically inaccessible AOVMs of the brain were treated using helium ion (47 patients) or linear accelerator (10 patients) radiosurgery. This study retrospectively evaluates the response of these AOVMs to treatment.All patients presented with previous hemorrhage. The mean patient age was 35.6 years (range, 13-71 yr). The mean AOVM volume was 2.25 cm3 (range, 0.080-15.2 cm3), treated with a mean of 18.0 Gy equivalent (physical dose x relative biological effectiveness, which is 1.3 for helium ion Bragg peak) (range, 7.0-40 Gy equivalent). The Drake scale scores before treatment were as follows: excellent (25 patients), good (26 patients), and poor (6 patients). The mean follow-up period was 7.5 years (range, 9 mo-13.8 yr).Eighteen patients (32%) bled symptomatically (20 hemorrhages) after radiosurgery. Sixteen hemorrhages occurred within 36 months after radiosurgery (9.4% annual bleed rate; 16 hemorrhages/171 patient yr); 4 hemorrhages occurred more than 36 months after treatment (1.6% annual bleed rate; 4 hemorrhages/257 patient yr) (P < 0.001). Complications included symptomatic radiation edema (four patients, 7%), necrosis (one patient, 2%), and increased seizure frequency (one patient, 2%). Eight patients underwent surgical resection of their AOVMs 8 to 59 months after radiosurgery because of subsequent hemorrhage. The Drake scale scores after treatment were as follows: excellent (25 patients), good (24 patients), poor (3 patients), and dead (5 patients, 3 of whom died as a result of causes unrelated to the AOVMs or radiosurgery).Radiosurgery may be useful for AOVMs located in surgically inaccessible regions of the brain. A significant decrease in bleed rate exists more than 3 years after treatment compared with the bleed rate within 3 years of treatment. Because current neuroradiological techniques are not able to image obliterative response in these slow-flow vascular lesions, longer term clinical follow-up is required.

Abstract

The goal of this study was to evaluate the pathological changes associated with radiation treatment (stereotactic radiosurgery or conventional irradiation) of angiographically occult vascular malformations (AOVMs).Eleven patients underwent surgical resection of an AOVM in the mesial temporal lobe, brain stem, thalamus, or basal ganglia after previous radiation treatment. The indications for surgery were recurrent symptomatic bleeding from the lesion in 10 patients and recurrent intractable seizures in 1 patient. Radiation was used as the initial therapy because the risk of surgical resection was deemed too high. Three patients received conventional radiation therapy of 3000 to 5400 rads at an outside institution. One patient received radiosurgery with the gamma knife at another institution using a dose of 15 Gy to the margin. The remaining 7 patients received stereotactic radiosurgery with a helium-ion particle beam. The dose range was from 18 to 26 Gy equivalents. The interval from radiation to surgical resection ranged from 1 to 10 years, with a mean of 3.5 years. These lesions were compared with 10 nonirradiated cavernous malformations.One irradiated lesion was identified pathologically as a true arteriovenous malformation despite being angiographically occult. This lesion did not demonstrate significant changes in the vasculature but did have radiation necrosis of the surrounding brain 5 years after 25 Gy equivalents of helium-ion radiosurgery. Two other specimens were too small to identify the type of vascular malformation adequately. Of the remaining eight malformations identified as cavernous malformations, six showed a combination of marked fibrosis of the vascular channels, fibrinoid necrosis, and ferrugination. However, the fibrinoid necrosis was the only finding unique to the irradiated lesions compared with nonirradiated controls. All the irradiated lesions still had patent vascular channels; none were completely thrombosed.Radiosurgery or conventional radiation therapy did not cause histologic vascular obliteration in intracranial AOVMs evaluated 1 to 10 years (mean 3.5 yr) after radiation delivery. It should be recognized that these patients are irradiation failures who may not be representative of all irradiated patients. However, recurrent bleeding from AOVMs may relate to poor radiation response in some patients.

Abstract

We have developed a TCR-based vaccine approach for the treatment of T cell malignancies. TCR genes were isolated from C6VL, a T cell tumor of C57BL/Ka origin. The transmembrane encoding domains of the TCR genes were replaced by sequences encoding for phosphatidylinositol-linked cell surface expression. A high expressing cell line was produced by transfection and amplification of the TCR genes. Large quantities of soluble native C6VL TCR-alphabeta protein was obtained by treating the high-expressing cells with a specific phospholipase and purifying the released TCR by affinity chromatography. Following vaccination with the TCR linked to keyhole limpet hemocyanin, specific anti-TCR humoral responses were induced. Both the carrier protein and an adjuvant were required for optimal responses. Hyperimmune serum from vaccinated mice reacted specifically with C6VL cells, and the immunizations did not affect the TCR repertoire, which suggested that the immune response was Id specific. The TCR-vaccinated mice were specifically protected from a lethal number of C6VL tumor cells. B cell-deficient mice were not protected by TCR vaccinations. Similarly, TCR-immunized mice depleted of CD8+ cells prior to tumor challenge were not protected. Thus, C6VL TCR vaccine effectively stimulated tumor protection, which depends on the presence of both B cells and CD8+ T cells.

Abstract

The idiotype (Id) of the Ig expressed on the surface of non-Hodgkin's lymphoma cells is a suitable target for immunotherapy. Indeed, treatment with monoclonal anti-Id antibodies (Abs) can induce long-lasting clinical remissions. However, some of the treated patients relapse with a tumor expressing Ig with point mutations in the idio recognized by the particular monoclonal antibody (MoAb). The alternative approach of active immunization with tumor Id can cure the disease in mice with established tumors and is now being studied in clinical trials. Here, we tested the hypothesis that active immunization with the idiotype would evoke a polyclonal immune response that would cover mutated tumor variants. As a test system, we chose the tumor from a patient who had achieved a complete remission after therapy with anti-Id Ab but subsequently relapsed with a mutated tumor variant no longer binding the treatment Ab. Mice were immunized with proteins and genetic vaccines derived from the original tumor, including (1) Id-keyhole limpet hemocyanin protein, (2) Id single-chain variable fragment (scFv) granulocyte-macrophage colony-stimulating factor (GM-CSF) protein, (3) DNA encoding the Id, and (4) an adenovirus encoding the Id. All immunized mice developed a specific immune response detecting tumor-derived Id proteins from the original tumor and from all tumor variants. We conclude that active immunization with tumor Id can induce a polyclonal immune response and therefore may prevent the escape of mutated tumor variants.

Abstract

To evaluate the safety, pharmacokinetics, and biologic effect of multiple doses of the chimeric anti-CD20 monoclonal antibody (mAb) IDEC-C2B8 in patients with relapsed B-cell lymphoma.Twenty patients with relapsed low-grade (n = 15) or intermediate-/high-grade (n = 5) lymphoma received weekly infusions times four of 125 mg/m2 (n = 3), 250 mg/m2 (n = 7), or 375 mg/m2 (n = 10) of IDEC-C2B8.Infusional side effects during the initial infusion were mainly grade I/II fever, asthenia, chills, nausea, rash, and urticaria. More serious events were rare. Peripheral-blood B cells were rapidly depleted and slowly recovered over 3 to 6 months. There was no change in mean immunoglobulin (Ig) levels. Antibody serum half-life (and maximum concentration [Cmax]) generally increased between the first and fourth infusions (33.2 hours v 76.6 hours, respectively) following the 375-mg/m2 doses. Six of 18 assessable patients had a partial remission (PR), with a median time to disease progression of 6.4 months (range, 3 to 21.7). Minor responses (MRs) were observed in five patients and progressive disease (PD) in seven. Tumor responses occurred in peripheral blood, bone marrow (BM), spleen, bulky lymph nodes, and extranodal sites, and in patients who had relapsed following high-dose myeloablative chemotherapy. Six of 14 patients (40%) with a low-grade histology responded. Four of six with bulky disease had a PR.IDEC-C2B8 chimeric anti-CD20 mAb therapy is well tolerated and has clinical activity in patients with relapsed B-cell lymphoma. The 375-mg/m2 dose has been selected for a phase II trial in patients with relapsed low-grade or follicular B-cell lymphoma.

Abstract

IDEC-C2B8 is a chimeric monoclonal antibody (MoAb) directed against the B-cell-specific antigen CD20 expressed on non-Hodgkin's lymphomas (NHL). The MoAb mediates complement and antibody-dependent cell-mediated cytotoxicity and has direct antiproliferative effects against malignant B-cell lines in vitro. Phase I trials of single doses up to 500 mg/m2 and 4 weekly doses of 375 mg/m2 showed clinical responses with no dose-limiting toxicity. We conducted a phase II, multicenter study evaluating four weekly infusions of 375 mg/m2 IDEC-C2B8 in patients with relapsed low-grade or follicular NHL (Working Formulation groups A-D). Patients were monitored for adverse events, antibody pharmacokinetics, and clinical response. Thirty-seven patients with a median age of 58 years (range, 29 to 81 years) were treated. All patients had relapsed after chemotherapy (median of 2 prior regimens) and 54% had failed aggressive chemotherapy. Infusional side effects (grade 1-2) consisting of mild fever, chills, respiratory symptoms, and occasionally hypotension were observed mostly with the initial antibody infusion and were rare with subsequent doses. Peripheral blood B-cell depletion occurred rapidly, with recovery beginning 6 months posttreatment. There were no significant changes in mean IgG levels and infections were not increased over what would be expected in this population. Clinical remissions were observed in 17 patients (3 complete remissions and 14 partial remissions), yielding an intent to treat response rate of 46%. The onset of these tumor responses was as soon as 1 month posttreatment and reached a maximum by 4 months posttreatment. In the 17 responders, the median time to progression was 10.2 months (5 patients exceeding 20 months). Likelihood of tumor response was associated with a follicular histology, with the ability to sustain a high serum level of antibody after the first infusion, and with a longer duration of remission to prior chemotherapy. One patient developed a detectable but not quantifiable immune response to the antibody that had no clinical significance. IDEC-C2B8 in a dose of 375 mg/m2 weekly for 4 weeks has antitumor activity in patients with relapsed low-grade or follicular NHL. Results with this brief, outpatient treatment compare favorably with results with standard chemotherapy, and IDEC-C2B8 has a better safety profile. Further studies evaluating IDEC-C2B8 in other types of lymphoma either alone or combined with chemotherapy are warranted.

Abstract

With conventional therapy, multiple myeloma (MM) has a poor prognosis. During the last few years, it has become clear that high-dose chemotherapy with autologous stem cell support can increase overall survival of MM patients, but further improvement in outcome is desperately needed. The monoclonal immunoglobulin (Ig) produced by the MM cells called idiotypes (Id) is a tumor-specific antigen due to unique antigenic determinants that are localized in the variable regions of the Ig molecule. Conceivably, Id immunization of MM patients in complete remission could further increase survival. Here we review the scientific basis for such Id immunization.

Abstract

The surface Ig on each B-cell lymphoma has unique portions (idiotypes), which can be recognized by the immune system. In this study, we immunized patients against the Ig expressed by their tumor and observed their clinical outcomes. After standard chemotherapy, 41 patients with non-Hodgkin's B-cell lymphoma received a series of injections with a vaccine consisting of tumor Ig protein coupled to keyhole limpet hemocyanin and emulsified in an immunologic adjuvant. Subjects were observed for toxicity, immune responses, and tumor status. The median duration of follow-up of all patients is 7.3 years from diagnosis and 5.3 years from the last chemotherapy given before vaccine treatment. Twenty patients (49%) generated specific immune responses against the idiotypes of their tumor Ig. Two patients who had residual disease experienced complete tumor regression in association with the development of these immune responses. The median duration of freedom from disease progression and overall survival of all 20 patients mounting an anti-idiotype immune response are significantly prolonged compared to the patients who did not mount an immune response. Thirty-two patients were in their first remission and nine were in subsequent remissions before beginning vaccine treatments. Analysis of the 32 first remission patients also shows an improved clinical outcome for those patients who mounted a specific immune response compared to those who did not (freedom from progression, 7.9 years v 1.3 years P = .0001; median survival from time of last chemotherapy not yet reached v 7 years, P = .04). This study confirms an earlier report that patients with B-cell lymphoma can be induced to make a specific immune response against the Ig expressed by their own tumor. It further shows that the ability to make such an immune response is correlated with a more favorable clinical outcome. Prospective controlled trials will be needed to prove a causal relationship between anti-idiotype immunity and improved clinical outcome.

Abstract

The idiotypic determinants of B cell lymphoma provide a tumor-specific Ag and a target for immunotherapy. We have developed several generations of idiotype vaccines that were tested in an animal model, the 38C13 mouse B cell lymphoma. Initially we showed that effective tumor immunity was elicited by the syngeneic Id when it was conjugated to a carrier protein and mixed with an adjuvant. A subsequent generation of Id vaccines eliminated the need for a carrier protein and for an adjuvant by incorporating cytokines into fusion proteins containing the Id. A third generation of vaccines consisting of naked DNA encoding the Id-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins was equally effective in inducing tumor immunity. To determine whether Ig variable regions, in the absence of constant regions, could be immunotherapeutic in this model, we tested the use of single-chain Fv (scFv). scFv proteins, produced in bacteria, and naked DNA encoding scFv were used in this study. scFv was tested alone or fused to GM-CSF or an immunoenhancing peptide derived from IL-1beta. Here we demonstrate that scFv-GM-CSF was effective only when injected as a protein, not as a DNA vaccine. In contrast, both scFv-IL-1beta peptide fusion protein and naked DNA encoding it induced tumor immunity that protected mice from tumor challenge.

Abstract

Idiotypic determinants of the immunoglobulin expressed on the surface of B-cell lymphomas are tumor-specific antigens (TSAs), which can be targeted by immunotherapy. Immunization with DNA constructs encoding the idiotype (ld) of a murine B-cell lymphoma induced specific anti-ld antibody responses and protected mice against tumor challenge. Use of DNA encoding an ld/GM-CSF (idiotype/granulocyte-macrophage colony-stimulating factor) fusion protein improved vaccine efficacy, and xenogeneic immunoglobulin constant region determinants were required for immunogenicity. These results indicate that DNA may be a simple and efficacious means of inducing immune responses against a weak, otherwise unrecognized tumor antigen, provided that additional stimuli are included with the DNA.

Abstract

Patients with non-Hodgkin's B-cell lymphoma who received an antitumor vaccine of idiotypic ig protein showed humoral and proliferative immune responses. Because immunity to some antigens, including tumor antigens and human pathogenic viruses, may be better correlated with the cytolytic cellular immune response, we evaluated 16 non-Hodgkin's lymphoma patients immunized with autologous idiotypic ig molecules for changes in tumor-specific cytotoxic T-lymphocyte precursor (CTLp) frequency using limiting dilution analysis. Eleven patients had a significant increase in tumor-specific CTLp. Eight of these 11 patients remain without evidence of disease or with stable minimal disease. In contrast, all five patients who did not have a significant change in tumor-specific CTLp have developed progressive disease. Patient vaccination with tumor associated protein antigens can increase tumor-specific CTLp frequencies. The correlation of increased tumor specific CTLp with freedom from progression is significant at P = .002. This study indicates that measurement of CTLp frequencies are relevant to the clinical evaluation of human tumor vaccines and suggests that cell-mediated cytolytic immune responses may be an important determinant of vaccine efficacy.

Abstract

Although radiosurgery is effective in obliterating small arteriovenous malformations (AVMs), it has a lower success rate for thrombosing larger AVMs. The authors surgically resected AVMs from 33 patients ranging in age from 7 to 64 years (mean 30.4 years) 1 to 11 years after radiosurgery. Initial AVM volumes were 0.8 to 117 cm3 (mean 21.6 cm3), and doses ranged from 4.6 to 45 GyE (mean 21.2 GyE). Of 27 AVMs in eloquent or critical areas, 10 were located in language, motor, sensory, or visual cortex, 11 in the basal ganglia/thalamus, one each in the brainstem, hypothalamus, and cerebellum, and three in the corpus callosum. Venous drainage was deep in 13, superficial in 12, or both in eight lesions. Spetzler-Martin grades were II in one, III in 12, IV in 16, and V in four patients. Eight patients experienced rebleeding after radiosurgery but prior to surgery. Three patients developed radiation necrosis and 25 underwent endovascular embolization prior to surgery. At surgery the AVMs were found to be markedly less vascular, partially thrombosed, and more easily resected, compared to those seen in patients who had not undergone radiosurgery. Pathological investigation showed endothelial proliferation with hyaline and calcium in vessel walls. There was partial or complete thrombosis of some AVM vessels and evidence of vessel and brain necrosis in many cases. Complete resection was achieved in 28 patients and partial resection in five. Clinical outcome was excellent or good in 31 cases, and two patients died of rebleeding from residual AVM. Four patients' conditions worsened following microsurgical resection. Final clinical outcome was largely related to the pretreatment grade. Radiosurgery several years prior to open microsurgery may prove to be a useful adjunct in treating unusually large and complex AVMs.

Abstract

A Phase I/II dose escalation study of 90Y-murine anti-CD20 monoclonal antibody (mAb) in patients with recurrent B-cell lymphoma was performed. The primary objectives of the study were: (a) to determine the effect of the preinfusion of unlabeled anti-CD20 mAb on the biodistribution of 111In-anti-CD20 mAb; (b) to determine the maximal tolerated dose of 90Y-anti-CD20 mAb that does not require bone marrow transplantation; and (c) to evaluate the safety and antitumor effect of 90Y-anti-CD20 mAb in patients with recurrent B-cell lymphoma. Eighteen patients with relapsed low- or intermediate-grade non-Hodgkin's lymphoma were treated. Biodistribution studies with 111In-anti-CD20 mAb were performed prior to therapy. Groups of three or four patients were treated at dose levels of approximately 13.5, 20, 30, 40, and 50 mCi 90Y-anti-CD20 mAb. Three patients were retreated at the 40-mCi dose level. The use of unlabeled antibody affected the biodistribution favorably. Nonhematological toxicity was minimal. The only significant toxicity was myelosuppression. The overall response rate following a single dose of 90Y-anti-CD20 mAb therapy was 72%, with six complete responses and seven partial responses and freedom from progression of 3-29+ months following treatment. Radioimmunotherapy with =50 mCi 90Y-anti-CD20 mAb resulted in minimal nonhematological toxicity and durable clinical responses in patients with recurrent B-cell lymphoma. Doses of =40 mCi 90Y-anti-CD20 mAb were not myeloablative.

Abstract

Chimeric anti-CD4 monoclonal antibody was administered intravenously as a single dose to eight patients with mycosis fungoides. The dose was escalated throughout the study between patients groups, and individual patients received 50, 100, or 200 mg per dose. Seven of eight patients responded to treatment with an average freedom from progression of 25 weeks (range, 6 to 52 weeks). The treatment was well tolerated, and there was no clinical evidence of immunosuppression. Following treatment, there was significant suppression of peripheral blood CD4 counts in all patients for 1 to 22+ weeks. Only one patient made a very low titer human antichimeric antibody response. All but two patients made primary antibody and T-cell proliferative responses to a foreign antigen administered 24 hours after antibody infusion. However, there was generally marked, but temporary suppression of T-cell proliferative responses in vitro to phytohemagglutinin (PHA), tetanus toxoid, and normal donor lymphocytes. We conclude that at the dose levels studied, this antibody (1) had clinical efficacy against mycosis fungoides; (2) was well tolerated; (3) had a low level of immunogenicity; (4) decreased T-cell proliferative responses in vitro, and (5) did not induce tolerance to a foreign antigen.

Abstract

In this pilot study, we investigated the ability of autologous dendritic cells pulsed ex vivo with tumor-specific idiotype protein to stimulate host antitumor immunity when infused as a vaccine. Four patients with follicular B-cell lymphoma received a series of three or four infusions of antigen-pulsed dendritic cells followed, in each instance, by subcutaneous injections of soluble antigen two weeks later. All patients developed measurable antitumor cellular immune responses. In addition, clinical responses have been measured with one patient experiencing complete tumor regression, a second patient having partial tumor regression, and a third patient resolving all evidence of disease as detected by a sensitive tumor-specific molecular analysis.

Abstract

Synthetic peptide ligands specific for the surface immunoglobulin receptor of the human Burkitt's lymphoma cell line SUP-B8, previously identified using phage display libraries, induced apoptosis of the SUP-B8 cells in vitro when administered as dimers or tetramers. The use of synthetic peptide ligands is being explored for immunotherapy of B-cell lymphoma. It will be critical to identify which of the peptide ligands identified are the most active functionally. Using the Cytosensor microphysiometer, SUP-B8 cells and B-lymphoma cells obtained from patients were found to acidify their extracellular environment within minutes of specific activation by surrogate peptide ligands or by anti-idiotype antibodies. This signal was blocked by pretreatment of the lymphoma cells with the tyrosine kinase inhibitor genistein. Treatment of SUP-B8 cells with dimeric and tetrameric specific peptide ligands caused a rapid increase in extracellular acidification rate, which peaked after 10 min at approximately 15 and 20% above basal rates, respectively. These responses were blocked by excess monomeric peptide. To evaluate the ability of different peptide ligands to induce a signal directly on lymphoma cells, thereby establishing their relative affinity to the surface immunoglobulin receptor, acidification rate changes were measured at varying peptide concentrations. The microphysiometer signal correlated with the known relative affinities and antiproliferative potencies of the peptides. This approach is particularly useful for primary tumor cells that cannot be cultured. The signal may be predictive of the efficacy of treatment with synthetic peptide ligands and may be useful in the evaluation of ligands for other cell surface receptors with biological effects on B-lymphoma cells.

Abstract

Ig heavy chain variable region (VH) genes expressed by human diffuse large-cell lymphoma (DLC) and follicular lymphoma (FL) were identified and analyzed with respect to germline gene families. In 67 cases of FL, VH region genes were expressed in a pattern similar to that of normal B cells, with a predominance of the large VH3 gene family being used. In contrast, of the 17 cases of DLC, there was an extremely biased use of VH genes. Of these DLC tumors, 88% expressed genes from the small VH4 gene family; and even among these tumors, there was a limited use of genes, with 11 cases producing Igs derived from the VH4.21 germline gene. Although most of the VH genes expressed by DLC tumor cells contained mutations with respect to their germline counterparts, almost all of these mutations occurred before the clonal expansion of the tumor. This contrasts with our previous findings of ongoing mutations in FL and represents a fundamental difference between these two malignancies. This preferential gene use implies an important role for the VH4 gene family, and specifically for VH4.21, in the genesis of DLC.

Abstract

Fusion proteins consisting of an Ig containing xenogenic constant regions and granulocyte-macrophage colony stimulating factor (Id-GM-CSF) are potent immunogens capable of inducing anti-idiotypic Abs after two immunizations, without the usual need for adjuvants or carrier proteins. In this study, we investigated the effects of hyperimmunization with Id-GM-CSF and found that it induces anti-GM-CSF Abs that could bind to GM-CSF and neutralize its bioactivity in vitro. However, no detrimental effects of the anti-GM-CSF activity were apparent on the general health of the animals or on their base line white blood cell counts. Mice with the anti-GM-CSF activity reconstituted their peripheral white blood cells with identical kinetics as control mice after high dose cyclophosphamide treatment, sublethal irradiation, or lethal irradiation followed by syngeneic bone marrow transplantation. Primary and secondary Ab responses to a variety of protein Ags, including an unrelated Ig Id, were not affected. However, the anti-Id response induced by an unrelated GM-CSF fusion protein that is dependent upon the GM-CSF bioactivity was impaired. To avoid any potential problems associated with inducing anti-GM-CSF Abs, we show that priming with the Id-GM-CSF protein and boosting with the Id protein alone were sufficient to induce comparable anti-Id titers without inducing anti-GM-CSF Abs. We conclude that although hyperimmunization of mice with the GM-CSF fusion protein induced neutralizing anti-GM-CSF Abs, this was of little consequence to the animals. Nevertheless, we have devised a strategy to overcome this potential limitation on the use of GM-CSF fusion proteins for immunization.

Abstract

Idiotypic determinants, antigenic sites expressed on the variable region of Ig molecules of malignant B cells, represent tumor-specific Ags but are weak immunogens. We have previously shown that the immunogenicity can be dramatically increased by fusing tumor Id to granulocyte macrophage (GM)-CSF. Here, we demonstrate that fusion proteins with IL-2 or IL-4 can also be highly immunogenic. Co-immunization of these fusion proteins with another Id demonstrated the importance of physical linkage between the cytokine and relevant Ag for this enhancement. All three fusion proteins are capable of eliciting significant levels of specific Abs against the Id without the use of carrier proteins or adjuvants, although the GM-CSF fusion protein appeared to be unique in its ability to induce higher titers of anti-Id Abs in the primary response. Furthermore, the Id-IL-2 fusion protein induced high titers of IgG2a and IgG3 anti-Id Abs, whereas the Id-IL-4 and Id-GM-CSF fusion proteins did not. Despite the differences, tumor protection was comparable in all mice having significant titers of anti-Id Abs, regardless of the fusion protein used. We concluded that Id-cytokine fusion proteins are potent immunogens that can elicit significant antitumor immunity. The general approach of fusing a cytokine to a potential Ag may be applicable to the design of vaccines for immunotherapy of other types of tumors as well as for other pathogens and disease states.

Abstract

The B-cell antigen CD20 is expressed on normal B cells and by nearly all B-cell lymphomas. This nonmodulating antigen provides an excellent target for antibody-directed therapies. A chimeric anti-CD20 antibody (IDEC-C2B8), consisting of human IgG1-kappa constant regions and variable regions from the murine monoclonal anti-CD20 antibody IDEC-2B8, has been produced for clinical trials. It lyses CD20+ cells in vitro via complement and antibody-dependent cell-mediated lysis. Preclinical studies have shown that the chimeric antibody selectively depletes B cells in blood and lymph nodes in macaque monkeys. In this phase I clinical trial, 15 patients (3 per dose level) with relapsed low-grade B-cell lymphoma were treated with a single dose (10, 50, 100, 250, or 500 mg/m2) of antibody administered intravenously. Treatment-related symptoms correlated with the number of circulating CD20 cells and grade II events consisted of fever (5 patients); nausea (2), rigor (2), orthostatic hypotension (2), bronchospasm (1), and thrombocytopenia (1). No significant toxicities were observed during the 3 months of follow-up. Serum C3, IgG, and IgM levels, neutrophils, and T cells were largely unchanged. At the three higher dose levels, pharmacokinetics of the free antibody showed a serum half-life of 4.4 days (range, 1.6 to 10.5). Levels greater than 10 micrograms/mL persisted in 6 of 9 patients for more than 14 days. No quantifiable immune responses to the infused antibody have been detected. CD20+ B cells were rapidly and specifically depleted in the peripheral blood at 24 to 72 hours and remained depleted for at least 2 to 3 months in most patients. Two-week postinfusion tumor biopsies showed the chimeric antibody bound to tumor cells and a decrease in the percentage of B cells. Tumor regressions occurred in 6 of 15 patients (2 partial and 4 minor responses). The results of this single-dose trial have been used to design a multiple-dose phase I/II study.

SYNTHETIC PEPTIDE LIGANDS OF THE ANTIGEN-BINDING RECEPTOR INDUCE PROGRAMMED CELL-DEATH IN A HUMAN B-CELL LYMPHOMAPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICARenschler, M. F., Bhatt, R. R., Dower, W. J., Levy, R.1994; 91 (9): 3623-3627

Abstract

Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

Abstract

Custom-made monoclonal anti-idiotype antibodies (anti-Id MoAbs) have been tested as a treatment modality in 34 non-Hodgkin's lymphoma (NHL) patients. Partial or complete tumor remissions have been induced with this treatment in 68% of these patients. One mechanism by which anti-idiotype antibodies may have induced these tumor responses is via a direct antiproliferative effect on the tumor cells, resulting in apoptosis. Primary NHL cells do not proliferate well enough in vitro to test this hypothesis directly. Therefore, we studied the effect of anti-idiotype antibodies on signal transduction through the surface Ig receptor as measured by the induction of cellular protein tyrosine phosphorylation. To assess whether bcl-2 protein could protect lymphoma cells from death induced by anti-Id MoAb, we also measured the level of bcl-2 protein in the same tumor cells. We found a strong correlation between the ability of an anti-Id MoAb to induce an increase in tyrosine phosphorylation in vitro and its ability to induce a tumor regression in the patient. By contrast, the level of bcl-2 expressed by the tumor cells was not correlated with clinical response to anti-Id MoAb treatment.

Abstract

It is well recognized that patients with classical mycosis fungoides (MF) may develop a large-cell lymphoma (LCL), a phenomenon known as "transformation." An unresolved issue regarding the transformation of MF is whether MF and LCL represent two separate lymphomas or whether they are derived from the same T-cell clone. We report the clinicopathologic, immunophenotypic, and immunogenotypic analysis of MF and LCL in a white male. He developed a rash at age 51 that was diagnosed at age 56 as clinical stage IA patch/plaque MF. After topical nitrogen mustard and total skin electron beam therapy for progressive generalized CD3+CD4+ patch/plaque lesions, he developed nodules of Ki-1+ (CD30+) T-LCL at age 72. Southern blot analysis of DNA digested with Bg/II or BamHI and probed with a T-cell receptor (TCR)-beta gene J beta 1/J beta 2 probe showed a single, identical rearranged band in both the MF and LCL skin lesions that had been obtained 4 years apart. V beta gene family--specific gene amplification assays demonstrated dominant V beta 6 PCR products in both types of lesions. These PCR products and lesional cDNA exhibited a monoclonal pattern when amplified with consensus TCR-beta gene VDJ joint primers and electrophoresed under conditions that allowed the resolution of small differences in size. Furthermore, sequence analysis of the V beta 6 PCR products amplified from both the MF and LCL lesions showed an identical nucleotide sequence involving V beta 6.4, D beta 1.1, J beta 1.2, and C beta 1. These findings indicate that both the MF and the LCL in this patient arose from the same T-cell clone and that these diseases developed at a stage in the clone's differentiation subsequent to rearrangement of the TCR-beta gene.

Abstract

To produce a vaccine against cancer, antigens must be found that are preferentially expressed by tumour cells and can induce an immune response against the tumour. The variable regions of the immunoglobulin molecules expressed on malignant B cells (idiotypes) are tumour-specific, but are weak immunogens. To induce an immune response in animals or humans, the idiotypic protein has therefore to be chemically coupled to a strongly immunogenic protein and mixed with an adjuvant. The resulting response can protect animals from subsequent tumour challenge, and cure animals with established tumours in combination with chemotherapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) augments antigen presentation in a variety of cells. Here we show that by fusing a tumour-derived idiotype to GM-CSF, it can be converted into a strong immunogen capable of inducing idiotype-specific antibodies without other carrier proteins or adjuvants and of protecting recipient animals from challenge with an otherwise lethal dose of tumour cells. This approach may be applicable to the design of vaccines for a variety of other diseases.

Abstract

Previous reports of embolization of cerebral arteriovenous malformations (AVMs) have evaluated the technique as adjunctive therapy prior to surgery or radiosurgery; our aim is to assess the role of embolization following radiosurgery.Six patients previously treated with radiosurgery and showing no response as judged by cerebral angiography were embolized 24 to 55 months (mean 34.3 months) after initial radiosurgery.In five of six, a significant volume reduction was achieved ranging from 60%-100% (mean 74%). One patient was treated with embolization alone and the AVM has remained fully thrombosed 2 years after treatment. Three patients underwent surgical resection for cure after embolization, and two patients had repeat radiosurgery to a significantly smaller AVM volume. One patient had an asymptomatic carotid dissection at embolization; however, no clinically apparent complications occurred in the treatment group.Embolization can be used after radiosurgery to assist in the management of those AVMs that have not responded to initial treatment.

Abstract

The t(14;18) chromosomal translocation characteristic of follicular lymphoma (FL) juxtaposes the immunoglobulin heavy chain locus (IGH) and the BCL2 proto-oncogene. The translocation can be readily detected as a non-germline Notl fragment resolved by pulsed-field gel electrophoresis. A benefit of this approach is that it enables examination of the structure of a large region (+/- 300 kb) surrounding the chromosomal breakpoint. In 40/46 cases the observed translocated Notl fragment was smaller than the 680-690 kb expected from published restriction maps of the involved loci suggesting a deletion in the region of the breakpoint. Analysis of the der(14) allele by molecular hybridization demonstrated that in 35/46 cases the mu constant region (C mu) was deleted. Further molecular dissection of the IGH locus demonstrated that this resulted from an interstitial deletion of the der(14) chromosome within the region defined by the mu switch region (S mu) on the 5' side and the epsilon constant region (C epsilon) on the 3' end. Thus, the deletion resembled a class switch (CS) recombination event. Surprisingly, the CS deletion was as common in FL which was sIGM positive (24/33, 72.7%) as in cases where the productive allele had already undergone CS deletion (11/13, 84.6%) suggesting that the observed non-physiologic CS deletion resulted from a cis effect of the chromosomal translocation. Similar interstitial deletions of the non-productive IGH allele were not seen in B cell lymphocytic lymphomas which do not have the t(14;18) translocation. Mapping of the 3' extent of the deletion by an isotype PCR assay demonstrated frequent involvement (11/12 cases) of the gamma 1 constant region (C gamma 1). Analysis of cases in which the deletion was not evident by Southern blotting but detectable by PCR suggested that the CS deletion had occurred in a small subpopulation of FL cells subsequent to the t(14;18) translocation. The biological role of frequent interstitial deletions of the der(14) chromosome in t(14;18)-carrying lymphomas remains to be elucidated.

Abstract

The authors evaluated the Coulter STKS (Coulter Corp., Hialeah, FL) five-part differential in a tertiary-care hospital using samples with a broad range of distributional and morphologic abnormalities. Particular attention was given to the performance of the instrument-generated suspect flags that occur as an aid to identify samples with abnormal leukocytes. A morphologically abnormal, or positive, blood smear was defined by the presence of any blasts, malignant lymphoid cells, grossly dysplastic neutrophils, nucleated red blood cells (nRBC), platelet clumps, or reactive lymphocytes of more than 5%. The presence of any white blood cell-related suspect flag, except for Immature Granulocyte/Bands (i.e., Blasts, Variant Lymph, NRBC, Platelet Clumps, Review Slide, or WBC*R), was considered to be a positive instrument result. The STKS showed excellent quantitative results for the WBC differential compared with the manual differential when these "morphologic abnormalities" were absent in a 400-cell manual differential or low in numbers (< or = 5%). Specificity of these non-immature granulocyte/band suspect flags was good, with a false-positive rate of only 11.7%. Overall sensitivity in 113 samples with morphologic abnormalities was 67.3%. Sensitivity to detection of > or = 1% abnormal WBCs or > or = 1 nRBC/100 WBCs (a subset of 78 samples) was 80.8%. Sensitivity to detection of more than 5% abnormal WBCs or more than 5 nRBC/100 WBCs (a subset of 53 samples) was 84.9%. The primary deficiency was the inability of the STKS to flag samples with lymphoma cells, lymphoid blasts, or more than 5% reactive lymphocytes.

Abstract

The CELL-DYN 3000 (Unipath Corp., Mountain View, CA) differential was evaluated in a tertiary care hospital using samples with a broad range of distributional and morphologic abnormalities. Particular attention was directed to the performance of the instrument-generated suspect flags that occur as an aid to identify samples with abnormal leukocytes, as well as the estimates of abnormal cells that are made by the instrument. The CELL-DYN 3000 showed excellent quantitative results for the white blood cell differential compared with a 400-cell manual differential, in which morphologic abnormalities were absent or occurred in low numbers (< or = 5%). Specificity of the BLAST, VARIANT LYMPH, NRBC, WBC, or DIFF suspect flags (with the requirement that the blast estimate and variant lymphocyte estimate by the instrument be > or = 1%) was 82.6%. Sensitivity of these flags to detection of more than 5% "abnormal" leukocytes (blasts, malignant lymphoid cells, grossly dysplastic neutrophils, nucleated red blood cells, or reactive lymphocytes) or significant platelet clumping was 81.6%. The primary deficiency was the inability of the CELL-DYN 3000 to flag samples with small numbers (< or = 5%) of nucleated erythrocytes, lymphoid blasts, or hairy cells, or more than 5% reactive lymphocytes. Specificity of the IG flag (with immature granulocyte estimate > or = 3%) for immature granulocytes (metamyelocytes, myelocytes, or promyelocytes) was 94.9%. Sensitivity of the immature granulocyte flag varied from 41.7% for identifying IG > or = 1% to 100% for the three samples with immature granulocytes > or = 3%. Calculation of sensitivity and specificity to varying percentages of bands showed poor flagging performance, with many false-positive and false-negative results at all levels.

Abstract

The idiotypic determinants of the surface immunoglobulin of a B-cell lymphoma can serve as a clonal tumor-specific marker, which may have implications for immunotherapy. We sought to determine whether idiotype-specific immune responses against this autologous antigen could be induced in patients with B-cell lymphoma.Nine patients were selected who had minimal residual disease or a complete remission after chemotherapy. Each received a series of subcutaneous injections of the immunoglobulin derived from his or her tumor cells (immunoglobulin-idiotype protein), which had been conjugated to a protein carrier and mixed with an immunologic adjuvant.In seven of the nine patients the injections induced sustained idiotype-specific immunologic responses of the humoral type (two patients), the cell-mediated type (four patients), or both (one patient). The use of an adjuvant was essential for these immune responses. The induced antibodies bound specifically to autologous immunoglobulin idiotype, inhibited the binding of murine monoclonal antiidiotype antibodies, and bound autologous tumor cells. Cell-mediated responses were demonstrated by the specific proliferation of immune peripheral-blood mononuclear cells to the soluble immunoglobulin-idiotype protein in vitro. The tumors of both of the patients with measurable disease regressed completely. Toxicity associated with the vaccine was minimal and consisted only of mild reactions at the site of intramuscular injection.These results demonstrate that autologous immunoglobulin idiotype can be formulated into an immunogenic, tumor-specific antigen in humans with B-cell lymphoma, and they provide the background for large-scale trials of active specific immunotherapy of this disease.

Abstract

Although surface immunoglobulin plays a central role in the differentiation and growth of normal B-cells, its role in the growth of human B-cell malignancies is largely a matter of conjecture. Human follicular lymphomas are attractive systems to study in part because they are clones of cells sharing many similarities with germinal center B-cells which are critically dependent on antigen selection for survival. Nucleotide sequence information was determined for the immunoglobulin heavy chain variable genes expressed by two cases of follicular lymphoma. In addition, the germ line variable gene counterparts were also cloned and sequenced from biopsy material obtained from both of these patients. Numerous mutations from germ line were present in the variable genes from both of these cases, many of which accumulated during expansion and growth of these lymphomas. Moreover, the mutations that accumulated during tumor expansion were distributed in a manner that almost certainly was dependent on positive selection presumably mediated by contact with an antigen. These data indicate that antigen selection is probably important for the growth and clonal evolution of follicular lymphomas.

Abstract

The genesis of human follicular lymphoma (FL) is a multistep process. The initial event is thought to be the chromosomal translocation t(14;18)(q32;q21) juxtaposing the bcl-2 proto-oncogene with the immunoglobulin (Ig) H chain locus joining segment (JH) as an error of D-J or V-D joining in the pre-B cell. However, FL is recognized clinically as a tumor of surface Ig (sIg)-positive B cells with morphologic and phenotypic similarities to the centrocyte of the secondary immune response. Thus, additional steps must be involved in the clonal expansion of the FL tumor cell beyond the activation of bcl-2 as a consequence of the t(14;18) translocation. Like the normal centrocyte, somatic mutations accumulate in the variable (V) genes of FL tumor B cells. To determine if clonal expansion of FL occurs before or after the development of the malignant follicle, we sought to examine the evolution of the FL V gene from its unmutated germline (GL) counterpart. To obtain the GL gene we first cloned the productively rearranged V gene of patient MT FL and obtained the clone rMTF. A hybridization probe derived from the 2.1-kb region upstream of the V gene in clone rMTF identified a single band in Southern blot hybridization of GL DNA. This probe was used to screen a size-selected library, and candidate GL V genes were isolated. Two identical clones, MTGL1 and 2, proved to have upstream regions (USRs) that were colinear with the USR of the rMTF. Thus, the MTGL clones represent the unmutated GL V genes, which were productively rearranged in the MT FL. Comparison of the GL V gene sequence to a consensus of MT FL V gene sequences revealed 42 mutations, demonstrating that malignant clonal expansion occurred subsequent to the activation of somatic mutation, presumably in the malignant follicle. Furthermore, the individual FL V gene sequences segregated into two distinct patterns of mutation. The major population represented 71% of the clones, and the minor population 29%. To investigate possible mechanisms for the parallel selection of distinct tumor cell populations, we analyzed the pattern of silent and replacement mutations within the V gene sequences. We found that in the framework regions (FRs) of both populations there were significantly fewer replacement changes than expected, suggesting that negative selective pressure was maintaining the structural integrity of the sIg. In contrast, the complementarity determining regions (CDRs), which make up the antigen binding domain of Ig, had an excess of replacement changes, suggesting positive selection for altered ligand binding.

Abstract

The Ig idiotype of B-cell lymphoma can be used as a tumor-specific target. Prior trials with monoclonal anti-idiotype antibodies alone and combined with alpha-interferon have shown significant antitumor activity. In some patients, idiotype-negative tumors emerged after treatment. In this trial, patients with relapsed non-Hodgkin's lymphoma were treated with two identical courses of monoclonal anti-idiotype anti-body therapy. Concurrent with the second course, at a time when idiotype-negative cells were suspected to be proliferating, a pulse dose of chlorambucil was administered. Tumor biopsies obtained before the first and second courses of treatment and at relapse were analyzed for idiotype expression and proliferation. Thirteen patients received 24 courses of antibody with minimal toxicity. Eleven had tumor regression, with 1 complete remission, 8 partial remissions, and 2 minor remissions, with freedom from progression lasting a median of 7 months in responding patients. Idiotype-negative tumor cells appeared in some relapse specimens despite the use of chlorambucil. In retrospect, this was not surprising because there was no increase in the proliferative rate of these tumors at the time the drug was used. Anti-idiotype antibodies continue to demonstrate antitumor activity against B-cell lymphoma with minimal toxicity. The mechanism of the effect is presumed to involve both direct antiproliferative effects of the antibody on the tumor cells as well as indirect, more long-lasting effects on the host. The addition of a mild chemotherapeutic agent in the dose and schedule used here to the second cycle of antibody therapy did not interfere with the antitumor effect, nor did it decrease the emergence of idiotype-negative cells.

CLONAL EVOLUTION OF A FOLLICULAR LYMPHOMA - EVIDENCE FOR ANTIGEN SELECTIONPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABahler, D. W., Levy, R.1992; 89 (15): 6770-6774

Abstract

The potential role antigens play in growth stimulation or in clonal selection of follicular lymphomas is unknown. To study this issue, we sequenced the immunoglobulin heavy chain variable region genes expressed by a follicular lymphoma from multiple biopsy specimens and also cloned and sequenced the corresponding germ-line variable gene from this patient. Comparison to the germ-line gene revealed numerous nucleotide substitutions in all of the lymphoma variable gene sequences. Some of the substitutions may have occurred in the nonmalignant precursor B cell that gave rise to this lymphoma because they were shared among all of the variable genes, but many of the mutations accumulated as the malignant clone expanded. The mutations were distributed in such a way that strongly suggested the majority of tumor cells had been positively selected through their antigen receptor. This was especially evident for the mutations that developed late in the clonal evolution of this lymphoma. These findings indicate that antigen stimulation may be involved in the growth of follicular lymphoma tumors.

Abstract

Lethally irradiated C3H/HeN mice reconstituted with normal syngeneic bone marrow survived significantly longer than unmanipulated control mice following challenge with a lethal dose of 38C13 lymphoma cells 2 to 3 weeks post-bone marrow transplantation (BMT). Although the magnitude of this effect was modest, it was highly reproducible. This resistance-producing effect of BMT could be enhanced by interleukin 2 administration and could be abrogated by anti-asialo-GM1 antiserum treatment of recipients. These findings are consistent with the hypothesis that cells with a natural killer phenotype are activated by BMT and can mediate tumor resistance. These studies provide a model to explore the cellular basis, independent of donor alloreactivity, of the graft antitumor effect of BMT observed in humans.

Abstract

More than one-half of adults with non-Hodgkin's B cell lymphomas present with low-grade follicular lymphomas. These tumor cells are found in close association with follicular T lymphocytes and dendritic cells, suggesting that the surrounding cells may play a role in the support of follicular tumors. Supernatants from activated human peripheral blood lymphocytes were found to promote the in vitro proliferation of follicular tumor cells. This effect was entirely due to interleukin 3 (IL-3), a factor generally thought to cause the growth and differentiation of immature hematopoietic cells. IL-3 receptors were detected on fresh isolates of all primary follicular cell tumors examined. These findings suggest that follicular cell tumors may be dependent in vivo on IL-3 and that therapies directed against IL-3, its receptor, or the T cells that produce it may be effective treatment for follicular lymphoma.

Abstract

We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.

Abstract

The expression of T-cell antigen receptor beta-chain variable genes (V beta) was evaluated in 28 cases of mycosis fungoides. A novel polymerase chain reaction (PCR) technique was used to associate expression of particular V beta genes with monoclonal T-cell populations. In addition, the same biopsies used for PCR analysis were also examined for reactivity with a panel of seven monoclonal antibodies that specifically recognized V beta proteins from four different families. Only three cases clearly stained with the antibodies, a result consistent with a diverse set of V beta genes being used. This was confirmed by PCR analysis, which indicated that V beta genes from many different families were expressed by these tumors. Preferential use of the V beta 8 family, which had been previously use of the V beta 8 family, which had been previously reported for this disease, was not evident among the cases analyzed.

Abstract

Persistence of the underlying malignancy remains the major obstacle limiting the success of high-dose chemoradiotherapy with autologous bone marrow transplantation (BMT) for non-Hodgkin's lymphomas. We used the 38C13 murine B-cell lymphoma model to explore the approach of transferring tumor antigen-specific immunity with syngeneic BM as a protective element. Mice serving as syngeneic marrow donors were twice immunized with tumor-derived surface Ig protein, the idiotype of which serves as a tumor-specific antigen, or with a control Ig of matched isotype. Naive lethally irradiated recipients reconstituted with marrow from immune donors showed serologic tumor idiotype-specific immunity, as well as protection against lethal tumor challenge. The immunoprotective effect of immune marrow was also shown in lethally irradiated recipients partially protected by specific immunization post-BMT. Combined donor and recipient immunization also replaced the requirement for the booster immunization of the donor. These results provide the rationale for active immunization with purified surface Ig from autologous tumor as an adjunct to autologous BMT in humans.

Abstract

Thirty-six randomly selected cases of low grade follicular lymphoma (FL) were analyzed for Ig heavy chain variable region (VH) gene expression. Assignment to one of the six human VH gene families (VH1 to VH6) was made with a polymerase chain reaction-based technique using family-specific leader primers. The frequency of VH family use in FL was found to be similar to that reported for normal peripheral blood lymphocytes and is therefore also roughly proportional to VH family size. To evaluate expression within an individual family, all of the lymphoma VH genes from the middle size VH4 family were sequenced and compared with previously published sequences. Of these eight lymphoma VH sequences, six were most closely related to just two of the 10 known functional VH4 germline genes. Nonrandom usage by FL of the JH3, JH4, and JH5 joining segments was also observed. Nucleotide sequences were also determined for 10 randomly selected lymphoma VH genes from the large VH3 family. With one possible exception, none of these lymphoma VH sequences appear to represent any of the VH3 genes that may be preferentially used in the fetal repertoire.

Abstract

The t(14;18) chromosomal translocation that results in the juxtaposition of the bcl-2 proto-oncogene with the heavy chain JH locus is a common cytogenetic abnormality in human lymphoma. In particular, it is seen in about 85% of follicular lymphoma (FL) and up to one-third of diffuse lymphomas (DL). The chromosome 18 breakpoints have been shown to cluster into two regions. The major breakpoint region (mbr) within the 3' untranslated region of the bcl-2 proto-oncogene accounts for approximately 60% of the cases and the minor cluster region (mcr) 30 kb 3' of bcl-2 accounts for approximately 25% of the breakpoints. Because of variability in the position of the breakpoint, detection of the t(14;18) by Southern blot analysis provides an important clonal marker for the tumor. However, conventional electrophoresis (CE) fails to detect the translocation in 15% to 25% of cases. We have applied pulsed-field gel electrophoresis (PFGE) to the detection of the t(14;18) in a series of lymphoma prospectively analyzed by CE, polymerase chain reaction (PCR), and cytogenetic analysis. PFGE readily detected t(14;18) rearrangements as indicated by comigration of bands detected with probes for the mbr region (chromosome 18) and the JH locus (chromosome 14). In a series of 40 patients with FL, this method proved to be the most comprehensive for detection of the translocation compared with standard methods; in fact, in one case only PFGE was able to detect the chromosomal rearrangement. Ten percent of the FL cases were negative by all methods tested. In a separate analysis of matched tissue specimens from cases of tumor progression of FL to diffuse lymphoma, PFGE detected a common t(14;18) rearrangement confirming a clonal origin in seven of seven cases, whereas CE detected a rearrangement in only three of seven cases. Overall, PFGE was able to detect a translocation in 8 of 12 cases that were negative by CE and four of eight negative by cytogenetic analysis. In conclusion, PFGE analysis is more comprehensive than CE, PCR, and cytogenetic analysis for the detection of the t(14;18) breakpoint in tissue biopsies of malignant lymphoma.

AN EPITOPE ON THE TRANSFERRIN RECEPTOR PREFERENTIALLY EXPOSED DURING TUMOR PROGRESSION IN HUMAN LYMPHOMA IS CLOSE TO THE LIGAND-BINDING SITEBLOODTakahashi, S., Esserman, L., Levy, R.1991; 77 (4): 826-832

Abstract

We have previously reported an anti-transferrin receptor antibody, Trump, which was originally selected for its ability to discriminate low- and high-grade lymphomas. This feature was distinct from the other anti-transferrin receptor antibodies such as OKT9. In the present study, further immunochemical analysis was performed to define the nature of the antigenic site recognized by the Trump antibody. Trump was found to block the binding of transferrin both to solubilized and to surface transferrin receptors; conversely, transferrin could block the binding of Trump only to surface transferrin receptors. Therefore, the epitope recognized by Trump is near but not identical to the transferrin binding site. Stimulation of peripheral blood lymphocytes with phytohemagglutinin induced both the OKT9 epitope and the Trump epitope, but 12-phorbol 13 myristate acetate induced only the OKT9 epitope. Growth of some cell lines was inhibited by Trump but not by OKT9. No structural difference was found between transferrin receptor molecules reactive with Trump and those reactive with OKT9. In support of these results, Trump was able to immunoprecipitate transferrin receptor molecules solubilized from low-grade follicular lymphoma cells even though it did not bind to the receptors exposed on the surface of these cells. These findings imply that low-grade lymphoma cells differ from high-grade lymphoma cells not in the structures of their transferrin receptors but in their exposure of the molecule on the cell surface.

Abstract

Stereotactic radiosurgery has proven to be an effective method of treating selected inaccessible or inoperable arteriovenous malformations (AVMs) of the brain. Radiation-induced obliteration of successfully-treated AVMs, however, occurs only after some latent period after treatment, depending on size, location, and dose. An experimental compartmental flow model is proposed to describe the hemodynamic alterations in the AVM as a result of the pathophysiological changes after radiosurgery, and to analyze temporal alterations in AVM blood flow rates and pressure gradients before complete obliteration. In representative small (low-flow, 150 ml/min) and large (high-flow, 440 ml/min) AVMs, it is found that increases in pressure gradients across certain vascular structures within the AVM occur during the normal course of radiation-induced flow decrease and AVM obliteration. The magnitude of these pressure alterations, however, may be within the normal physiological variations in cerebrovascular blood pressure. The effects of partial-volume irradiation of the AVM is examined by limiting radiosurgical treatment to varying portions of the flow compartments within the model. It is found that alterations in pressure gradients persist in unirradiated vascular shunts, even after complete obliteration of the treated AVM volume. These pressure alterations may increase the probability of hemorrhage from the untreated shunts of the AVM and cause redistribution of regional cerebral blood flow resulting in increased flow through these untreated shunts.

Abstract

To investigate the clonal relationship between follicular lymphoma (FL) and transformed diffuse lymphoma (tDL), we examined the expression of tumor idiotype, immunoglobulin (Ig) gene rearrangements and sequence of Ig variable genes in paired tissue specimens. All 16 cases analyzed expressed surface immunoglobulin (sIg) on both the FL and the tDL, though the immunophenotype of one case of FL could not be definitively determined. In 14 of 15 cases, the surface immunophenotype was preserved; the exception was likely secondary to a class switch from IgM to IgG. In 12 of 13 cases, antiidiotypic monoclonal antibodies prepared against the FL reacted with the paired tDL. Analysis of Ig gene rearrangements in four cases by Southern blot hybridization showed evidence of clonal relationships in all cases though concordance was not seen with all probes tested (C kappa, C lambda, JH, PFL1, and PFL2). In the one case that had a discordant L chain rearrangement, sequence analysis of the L chain demonstrated a common mature B cell origin for both the FL and tDL. To determine whether tDL arose from one or more FL cells, the sequences of the H chain variable genes were analyzed. Individual clones of the V region gene of the FL showed a random distribution of changes throughout the sequence. In contrast, individual clones of the V region gene from tDL shared numerous nonrandom sequence alterations, implying a common single cell origin. In conclusion, tDL is a mature B cell and arises by transformation of a single FL cell.

Abstract

Studies in the 38C13 model, a lethal murine B-cell lymphoma of C3H origin, have previously demonstrated the efficacy of immunization with tumor idiotype against established tumors, especially in the setting of reduced tumor burden when combined with chemotherapy. We have extended these studies to test the protective effect of immunization with 38C13 idiotype protein (38C-Id), coupled to KLH and administered with an adjuvant, against a subsequent tumor challenge following lethal total body irradiation (950 R) and reconstitution with syngeneic bone marrow (20 x 10(6) cells) from normal donors. Animals prepared in this manner which were immunized with 38C-Id after 3 weeks recuperation and challenged with 1000 38C13 tumor cells 2 weeks later demonstrated significantly longer survival when compared to control animals which had been immunized with irrelevant idiotype protein. Irradiated reconstituted mice immunized after 5 weeks recuperation and challenged with 1000 tumor cells also demonstrated prolonged survival compared to controls, as well as a small number of cures (approximately 40%). Anti-38C-Id antibodies, implicated in the mechanism of idiotype induced anti-tumor immunity in this model, were detectable after immunization at both 3 and 5 weeks, although there was no significant correlation between serum antibody levels and survival of individual mice. These results suggest that immunologic recovery as early as 3 to 5 weeks following marrow grafting is sufficient to allow induction of idiotype-specific, anti-tumor immunity and form a model for our clinical trial of tumor idiotype vaccination for patients with B-cell lymphoma undergoing autologous BMT.

Abstract

Chimeric (murine/human) anti-CD4 monoclonal antibody was infused into seven patients with mycosis fungoides. Successive patients received doses of 10, 20, 40, and 80 mg of antibody twice a week for 3 consecutive weeks. All patients had some clinical improvement, but responses were of relatively short duration. Serum levels of chimeric antibody varied as a function of dose. At the 80-mg dose level, antibody was readily observed in biopsied skin lesions. Although there was coating by antibody of most CD4 positive cells in the blood, there was no significant depletion of CD4 positive cells. Low-level antibody responses against the mouse Ig variable region and human Ig allotypic constant region determinants were observed in several patients, but none were of clinical significance. All but two patients made primary antibody and T-cell proliferative responses to a simultaneously administered foreign protein test antigen. However, there was marked suppression of the mixed lymphocyte reaction. We conclude that at the dose levels studied, a chimeric anti-CD4 monoclonal antibody (1) had some clinical efficacy against mycosis fungoides; (2) was well tolerated; (3) had a low level of immunogenicity; (4) had immediate immunosuppressive effects; and (5) did not induce tolerance to a co-injected antigen.

Abstract

Recurrence of the underlying malignancy remains a major cause of treatment failure after autologous bone marrow transplantation (BMT) for patients with lymphoma. In this regard, we have developed an immunotherapeutic approach designed to induce resistance against residual tumor cells persisting after BMT. Previous studies in the model system of 38C13, a lethal B-cell lymphoma of C3H origin, have shown that active immunization with purified tumor-derived surface immunoglobulin (Id), as a tumor-associated antigen, produces resistance to tumor growth. Id immunization of lethally irradiated mice at 3 or 5 weeks after reconstitution with syngeneic bone marrow resulted in significantly prolonged survival after tumor challenge compared with nonspecifically immunized controls. Low levels of idiotype-specific antibody were also demonstrated in the sera of specifically immunized mice at this early time, when other functional studies in the literature of immunocompetence after syngeneic reconstitution might have predicted incomplete recovery. Immunization of mice before lethal irradiation and syngeneic marrow reconstitution also induced significant resistance to tumor challenge, suggesting the persistence of established host antitumor immunity through total body irradiation. These studies demonstrate the feasibility of id immunization in conjunction with bone marrow transplantation.

TAPA-1, THE TARGET OF AN ANTIPROLIFERATIVE ANTIBODY, IS ASSOCIATED ON THE CELL-SURFACE WITH THE LEU-13 ANTIGENJOURNAL OF IMMUNOLOGYTakahashi, S., Doss, C., Levy, S., Levy, R.1990; 145 (7): 2207-2213

Abstract

A murine mAb, 5A6 (IgG1), has been isolated by immunization with a human B lymphoma cell line and screening for growth inhibition. The antibody immunoprecipitated a single chain protein of 26 kDa from cell lysates made with Triton X-100 but additional proteins were precipitated when cell lysates were made with the milder detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio)-1-propane sulfate). We have identified one of these coprecipitated molecules as the 16-kDa Leu-13 Ag. 5A6 and anti-Leu-13 showed similar, although not identical, reactivity, growth inhibition and temperature-dependent aggregation effects among hematolymphoid cell lines. The aggregation induced by 5A6 and anti-Leu-13 was not dependent on LFA-1 (lymphocyte function-associated Ag-1). The cell-surface expression of both TAPA-1 (target of an antiproliferative antibody-1) and Leu-13 could be down-modulated by binding to their respective antibodies and they could be reciprocally comodulated. These results suggest that TAPA-1 and Leu-13 form a complex on the cell surface and play a role in growth control through a common pathway.

Abstract

The murine B-cell lymphoma 38C13 model was used to study the radiobiological effect of 131I-monoclonal antibody (MAB) therapy compared with dose equivalent external beam irradiation. Continuous exponentially decreasing low dose rate (LDR) gamma-irradiation, and multiply fractionated (MF) X-irradiation were compared with dose equivalent 131I-MAB. The relative therapeutic efficacy of radioimmunotherapy, and the relative contribution of (a) low dose rate; (b) whole body irradiation; and (c) microdosimetry to the overall effect were determined. Groups of mice with or without B-cell lymphoma were treated with either (a) 131I-anti-idiotype MAB; (b) 131I-isotype-matched irrelevant control MAB; (c) 5-15 Gy 250 kV X-irradiation given as a single fraction; (d) 2.5-30 Gy 250 kV X-irradiation given in 10 fractions/2 weeks; or by (e) continuous exponentially decreasing gamma-irradiation via a 137Cs source, which simulated the effective t1/2 of the 131I-MAB. In tumor-free mice the LD50/30 was approximately 10 Gy for MF and LDR external irradiation, and 11-12 Gy for 131I-MAB. However, the effect of these modes of irradiation on tumor size differed significantly. The cumulative percentage of tumor reduction averaged over 12 days was 0.635 +/- 0.055%/Gy for MF, and 1.36 +/- 0.061%/Gy for LDR external irradiation (a relative efficacy factor of 1.63 for LDR irradiation; P = 0.01). Assuming homogeneous body distribution, the tumor reduction effect over 12 days for 131I-MAB was 2.064 +/- 0.133%/Gy for specific, and 1.742 +/- 0.1%/Gy for nonspecific isotype-matched irrelevant 131I-MAB (P = 0.02). When 131I-MAB was compared to LDR external irradiation, the relative efficacy factor was 1.99 (P less than 0.001). In summary, there was a dose rate effect on tumor response, which may in part explain the efficacy of radioimmunotherapy. The additional effect of 131I-MAB on tumor response was only partially explained by the cumulative concentration ratio of 131I-MAB tumor/131I-MAB whole body, which was on average 1.7. This relatively low concentration ratio was partly due to tumor-mediated dehalogenation. Thus, the overall tumor response was a function of the total dose, dose rate, and both the specific and nonspecific distribution of 131I-MAB.

Abstract

A murine monoclonal antibody was identified by its ability to induce a reversible antiproliferative effect on a human lymphoma cell line. Immunoprecipitation studies revealed that the antibody reacted with a 26-kilodalton cell surface protein (TAPA-1). A diverse group of human cell lines, including hematolymphoid, neuroectodermal, and mesenchymal cells, expressed the TAPA-1 protein. Many of the lymphoid cell lines, in particular those derived from large cell lymphomas, were susceptible to the antiproliferative effects of the antibody. TAPA-1 may therefore play an important role in the regulation of lymphoma cell growth. A cDNA clone coding for TAPA-1 was isolated by using the monoclonal antibody to screen an expression library in COS cells. Analysis of the deduced amino acid sequence indicated that the protein is highly hydrophobic and that it contains four putative transmembrane domains and a potential N-myristoylation site. TAPA-1 showed strong homology with the CD37 leukocyte antigen and with the ME491 melanoma-associated antigen, both of which have been implicated in the regulation of cell growth.

Abstract

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.

Abstract

Heavy-charged-particle radiation has several advantages over protons and photons for the treatment of intracranial lesions; it has an improved physical distribution of the dose deep in tissue, a small angle of lateral scattering, and a sharp distal falloff of the dose.We present detailed clinical and radiologic follow-up in 86 patients with symptomatic but surgically inaccessible cerebral arteriovenous malformations that were treated with stereotactic helium-ion Bragg-peak radiation. The doses ranged from 8.8 to 34.6 Gy delivered to volumes of tissue of 0.3 to 70 cm3.Two years after radiation treatment, the rate of complete obliteration of the lesions, as detected angiographically, was 94 percent for lesions smaller than 4 cm3, 75 percent for those of 4 to 25 cm3, and 39 percent for those larger than 25 cm3. After three years, the rates of obliteration were 100, 95, and 70 percent, respectively. Major neurologic complications occurred in 10 patients (12 percent), of whom 8 had permanent deficits. All these complications occurred in the initial stage of the protocol, before the maximal dose of radiation was reduced to 19.2 Gy. In addition, hemorrhage occurred in 10 patients from residual malformations between 4 and 34 months after treatment. Seizures and headaches were less severe in 63 percent of the 35 and 68 percent of the 40 patients, respectively, who had them initially.Given the natural history of these inaccessible lesions and the high risks of surgery, we conclude that heavy-charged-particle radiation is an effective therapy for symptomatic, surgically inaccessible intracranial arteriovenous malformations. The current procedure has two disadvantages: a prolonged latency period before complete obliteration of the vascular lesion and a small risk of serious neurologic complications.

Abstract

To increase the efficiency of directionally cloning cDNA, we have constructed a pair of vectors and devised a cDNA cloning strategy that improves upon previously published methods. The vectors, pLIB: AZ and pLIB: ZA, have two unique (distinct religation specificities; GGCCN/NNNNGGCC) SfiI sites (SfiI.A and SfiI.B) flanking a stuffer fragment which contains the tetracycline-resistance element. These vectors permit the directional cloning of cDNA in both sense (pLIB: AZ) and antisense (pLIB: ZA) orientations relative to the promoter for phage T3 RNA polymerase. cDNA that was synthesized using a primer with a 5' sequence of a SfiI.B site followed by an oligo(dT)16 3' tail was then ligated to an adaptor with the sequence of a SfiI.A site produced directional molecules that could be cloned into the pLIB vectors. Complex libraries with 10(7) members were produced from as few as 6 x 10(5) cells. The SfiI sites and stuffer can be subcloned as a cassette to permit directional cloning in other vectors, as there are several restriction enzyme sites flanking this region to the 5' and 3'.

Abstract

We examined stimuli which are required for the induction of in vitro proliferation of follicular lymphoma cells, a low grade non-Hodgkin's B cell lymphoma characterized by a specific chromosomal translocation, t(14;18)(q32;q21), and by in vivo growth of the lymphoma cells in germinal center-like follicles infiltrated with CD4+ T cells. The purified follicular lymphoma cells, which are morphologically uniform, small, and dense, did not respond to stimulation with soluble lymphokines in the absence of T cells. Vigorous in vitro proliferation of follicular lymphoma cells was induced, however, when the follicular lymphoma cells were cultured with a CD4+ T cell clone which recognized alloantigens expressed by the lymphoma cells. This response required B-T cell contact, and was inhibited by anti-class II but not by anti-class I MHC mAb, indicating that these neoplastic B cells behaved as normal B cells and responded to normal activation and differentiation signals from T cells. After the cognate B lymphoma-T cell interaction occurred in culture, addition of IL-2 or IL-4 enhanced the proliferation of the tumor cells. These results, with a monoclonal and homogeneous population of B cells, affirm the idea that cognate interaction between B cells and Th cells is required for the effective activation of resting B cells. Moreover, these results suggest that a critical host-tumor interaction occurs in vivo, and that the polyclonal CD4+ T cells that infiltrate follicular lymphomas play a role in sustaining rather than inhibiting tumor growth in vivo. If so, therapies directed not only against the neoplastic cell but also against specific T cells and their cognate interactions with tumor cells may have a rationale.

Abstract

The Ig Id of a B cell lymphoma serves as a distinct marker of the malignant clone and thus as a tumor-specific target for antibody therapy. Somatic variation of the Ig genes expressed by B cell tumors can lead to loss of reactivity with anti-Id antibodies and escape of tumors from the therapeutic effects of such antibodies. In our study, we have used anti-Id antibodies to screen for variants within a cell line derived from a patient with a large cell lymphoma of the B cell type. Cells were simultaneously stained on their surface for idiotypic and for isotypic Ig determinants using reagents labeled with different fluorochromes. Tumor cells expressing intact Ig molecules with alteration of their idiotypic determinants were isolated with the fluorescence activated cell sorter. Idiotypic variation was an ongoing process in vitro with Id- variants being generated at a rate of 2.7 x 10(-4)/cell per generation and Ig- cells being produced at a rate of 1.31 x 10(-5)/cell per generation. Subcloned variants expressed subtle differences in reactivity with a panel of three non-cross-blocking anti-Id antibodies. Analysis of Ig gene rearrangements by the Southern blotting technique using a JH probe established that the variants and the original tumor cells were all clonally related. Immunoprecipitation of surface labeled Ig molecules from the variant subclones disclosed major alterations of the lambda-L chains with no gross alterations of the mu-H chains. Related studies have established that the tumor cells undergo rearrangement and expression of new lambda-L chain genes.

Abstract

Anti-idiotype monoclonal antibodies are now available for up to one-third of all patients with B-cell cancer. This is because some antibodies made in the past for individual patients cross-react with the idiotype expressed by other patients' tumor cells. Clinical trials with anti-idiotype antibodies have demonstrated reproducible antitumor effects in patients who have failed conventional treatments. The anti-idiotype antibody treatments are not associated with any significant toxicity and rarely induce immune responses in patients with B-cell lymphoma. They can therefore be used repetitively. Future developments will include the combination of anti-idiotype antibodies with other biologic therapies and with chemotherapy. In addition, one may be able to induce the patient to make an active immune response against the idiotype expressed by his or her tumor cells.

AN EPITOPE OF THE TRANSFERRIN RECEPTOR IS EXPOSED ON THE CELL-SURFACE OF HIGH-GRADE BUT NOT LOW-GRADE HUMAN LYMPHOMASBLOODEsserman, L., Takahashi, S., Rojas, V., Warnke, R., Levy, R.1989; 74 (8): 2718-2729

Abstract

In attempting to identify antigens that are differentially expressed on tumor cells following transformation from follicular small cleaved cell lymphoma (FSC) to immunoblastic lymphoma (IL), we identified a unique epitope of the transferrin receptor (TfR). The epitope is available for binding in aggressive lymphomas but not in indolent lymphomas or normal cells. An immunoglobulin G2a (IgG2a) antibody that binds this epitope, Trump, was produced by screening on tumor cells from a patient who initially had a low-grade lymphoma which subsequently converted to a high-grade lymphoma. Immunoprecipitation and comodulation studies show that Trump binds to the TfR, but blocking studies and immunostaining reveal that the TfR epitope seen by Trump is distinct from the OKT9 and anti-TfR binding sites. The ability of Trump to discriminate a separate population of more highly malignant cells suggests that the expression of the Trump epitope is determined by the state of activation or degree of malignancy of the cell. In addition, it may be possible to use the Trump antibody diagnostically or therapeutically in the management of lymphomas.

Abstract

An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

Abstract

A murine B cell lymphoma (38C13) was subjected to immunoselection with mAbs directed against the idiotypic determinants of its cell surface Ig. Variants emerged with altered Ig receptors containing identical heavy chains but different light chains. The functional light chain genes in these variants were composed of V kappa segments drawn from the V kappa Ox-1 family, which had replaced the V kappa gene expressed by the parental tumor by rearranging to downstream J kappa segments. Rearrangement at the kappa locus continued to occur spontaneously, giving rise to secondary and tertiary variants at a rate of 1.9 x 10(-4) per cell per generation. Variants were isolated that had ceased production of surface Ig but went on to rearrange again and to become surface Ig+. The Ig- state may be an intermediate step providing a stimulus for continued rearrangement. This process provides an additional mechanism for generating diversity within B cell clones and expands the use of the available repertoire of Ig genes.

Abstract

CTL are thought to play a role in the elimination of transformed cells in vivo. The effectiveness of such CTL is in part dependent on recognition of tumor specific antigens. Among the best characterized tumor-specific antigens are the unique or idiotypic determinants on the Ig of B cell lymphomas. Here we describe the generation and properties of human CTL specific for the idiotype on autologous B cell tumors. These cells are CD3+,CD4-,CD8- and express the delta chain of the TCR. Such cells may prove useful in tumor-specific adoptive therapy.

Abstract

Mature B cells that express surface immunoglobulin (Ig) are usually committed to their original Ig product. It was shown that such a cell can replace its light chain by rearranging and expressing a new light chain from the other allele. Anti-idiotype antibodies were used to isolate idiotypic variants from a surface IgM+lambda+ human B cell tumor line. The variants expressed a new lambda light chain. Both the original and the new lambda transcripts were present in the variant cells, but only the new one was expressed as a protein on the cell surface. Therefore, although the cell exhibited allelic exclusion and had only one Ig receptor at a time, the commitment to a particular light chain gene was reversible.

Abstract

Idiotypes are distinct clonal markers for B-cell lymphomas. Previously we reported the use of anti-idiotype antibodies in the therapy of patients with B-cell malignancies. Because synergy was demonstrated with the addition of alpha interferon to anti-idiotype antibodies in a murine lymphoma model, we performed a clinical trial combining these two agents. Here we provide an update of the original trial of anti-idiotype antibodies alone and report the outcome of the new combination trial. In 16 treatment courses of anti-idiotype antibodies alone there were seven partial responses and one complete response. In 12 courses of combination anti-idiotype antibody and alpha interferon there were two complete responses and seven partial responses. Substantial tumor regressions occurred with minimal toxicity in both trials even in patients refractory to conventional chemotherapy. Tumor specimens obtained at the time of disease progression often contained a preponderance of idiotype-negative lymphoma cells, suggesting that anti-idiotype antibody treatment exerted a strong antitumor effect against antigen-positive cells. Anti-idiotype antibodies have reproducible objective antitumor activity in B-cell lymphoma. The addition of alpha interferon may improve the initial rate of response to this treatment. Strategies that deal effectively with idiotype-negative lymphoma cells should improve the extent and duration of these responses.

Abstract

Previously, we have shown that a human T acute lymphoblastic leukemia cell line, HPB-ALL, exhibits clonal heterogeneity within its Ag receptor, as revealed by varying reactivity patterns with a panel of anti-idiotype mAb. We now extend these findings to another human T acute lymphoblastic leukemia cell line, SUP-T13, and to two fresh human chronic lymphocytic leukemias, JE and EF. In the two cell lines, two types of Ag receptor variants could be found: those that retained a receptor molecule but lost reactivity with an anti-idiotype mAb (idiotype variants), and those which had lost surface receptor expression completely (receptor-negative variants). The idiotype variants, at least in HPB-ALL, have differentially glycosylated receptor alpha-chains from the parent. The receptor-negative cells, in HPB-ALL as well as in SUP-T13, produce cytoplasmic receptor and CD3 proteins but do not transport them to the surface. Neither idiotype nor receptor-negative variants could be detected in either of the fresh tumors of chronic lymphocytic leukemias. The limit of sensitivity in these analyses was about 0.05%. We conclude that antigen receptor variants can spontaneously occur in cell lines derived from acute lymphoblastic leukemias, but are infrequent in chronic lymphocytic leukemias in vivo, and that therapy with anti-idiotype mAb may be a viable strategy for these malignancies.

Abstract

A murine B cell lymphoma (38C13) was used to study the efficacy of idiotype immunotherapy against established tumors. Immunization of mice with 38C13 tumor-derived Ig, administered after a lethal tumor inoculation, significantly prolonged survival of animals compared to control groups. The efficacy of active immunotherapy was dramatically enhanced when combined with chemotherapy. Cyclophosphamide (100 mg/kg), administered in combination with idiotype immunization to mice bearing 10-day-old, 1 to 2 cm diameter s.c. tumors, resulted in a significant prolongation of survival as compared with either cyclophosphamide or immunization alone and yielded approximately 50% cures. Additional studies combining active immunotherapy with surgical excision of the primary s.c. tumor nodule were less effective than combination chemoimmunotherapy, indicating that reduction of tumor burden was necessary, but not sufficient for effective treatment of established 38C13 lymphoma.

Abstract

Idiotype variants of 38C13, a murine B cell lymphoma, have been isolated by immunoselection with antiidiotype mAbs. The V region genes for the kappa light chains and mu heavy chains expressed by these tumor cells were sequenced and compared. There was no evidence for V region somatic point mutation in this tumor. However, while the heavy chain genes were all identical, the light chain genes were all different. The light chain genes of each variant were derived from the V kappa-Ox1 gene family and joined to J kappa 4, whereas the light chain gene of the parental tumor was derived from the V kappa 9 family and joined to J kappa 2. Two of the variants used the identical V kappa gene but differed by the inclusion of a variable number of additional nucleotides in the V/J joint. Thus, the idiotypic heterogeneity of this B cell lymphoma arises as a consequence of alternative light chain rearrangements rather than point mutation. This process repetitively uses members of the same V kappa gene family. Two of the variants use the identical V kappa and J kappa gene segments but differ by the presence of extra nucleotides at the V kappa/J kappa joint.

Abstract

We have sought to address the question of clonal variation of TCR within a human T leukemia cell line, HPB-ALL. To do so, a panel of anti-idiotypic antibodies was produced and the cell line examined for variants. We isolated both spontaneous idiotype and receptor-negative variants without applying mutagens or any selective pressure other than sorting the cells. These sorted and cloned populations are all clonally related to each other as shown by their beta-TCR locus gene rearrangements. The idiotype variants have alpha-chains which are differentially glycosylated, but they have the same size core protein after treatment with peptide N-glycosidase F to remove their carbohydrate side chains. This probably accounts for their idiotypic difference, since the antibody that distinguishes them appears dependent upon glycosylation for its binding, as shown by immunoprecipitation in the presence versus the absence of tunicamycin, which inhibits glycosylation from occurring. The idiotype variants differed from one another in variable region sequences by only a single amino acid substitution in the beta-chain, which is likely not important for the idiotypic difference. The receptor-negative variant produces both alpha- and beta-mRNA and cytoplasmic protein for TCR, but fails to transport this protein to the cell surface. We conclude that idiotype and receptor-negative variants of a T cell clone can occur in the absence of appreciable somatic mutation.

Abstract

Combination therapy with syngeneic anti-idiotype antibody and human hybrid rIFN-alpha A/D synergistically increase survival in C3H/HeN mice challenged with a lethal dose of tumor cells. C3H/HeJ mice, which have previously been described to be LPS hyporesponsive and have a defect in Fc gamma R function, did not respond to anti-idiotype therapy as well as C3H/HeN normal mice. This defect was completely corrected in animals treated simultaneously with IFN. Anti-idiotype mAb that was cleaved into F(ab')2 fragments no longer had any antitumor activity alone and could not be enhanced by IFN therapy. These results suggest that antibody is functioning through Fc gamma R-bearing effector cells that are enhanced by IFN therapy. Synergy between IFN and anti-idiotype mAb was maintained in nude mice lacking classical T cells but was reduced in C3H beige mice lacking classical NK/killer cells. IFN did not increase idiotype expression on the tumor cells but did increase H-2 expression. Although we have previously shown that rIFN-alpha A/D can directly kill 38C13 in vitro, an IFN-resistant subclone derived from 38C13, SIR-1, was equally or more responsive to human rIFN-alpha A/D in vivo and had a synergistic antitumor response to combination IFN and anti-idiotype therapy, indicating that IFN acts primarily through host mediated effects rather than direct effects.

Abstract

The genes coding for the Ig light chains expressed in two cases of human follicular lymphoma were cloned and sequenced. In each case, multiple independent isolates of the tumor population were compared. Although each tumor represented a single clone of B cells with a unique V/J joint, different cells within each tumor had accumulated multiple point mutations in the V gene during clonal expansion. Most of the mutations observed were silent, but some resulted in amino acid replacements. Identical silent mutations were often observed in independent isolates of each tumor. By combining the current data with VH sequences obtained previously from the same cells, it was apparent that the repetitive silent mutations could not be explained solely by a genealogic tree. Such mutations could represent hot spots whose tendency to mutate may be influenced by neighboring DNA sequences or by the methylation of specific cytosine residues.

Abstract

Two murine models, C3H 38C13 B-cell lymphoma and AKR SL2 T-cell lymphoma were used to determine the efficacy of three different interferon preparations, recombinant human hybrid interferon-alpha A/D, recombinant murine interferon (rMIFN)-gamma, and natural MIFN-alpha/beta (greater than or equal to 85% beta), alone and in combination with tumor specific and nonspecific monoclonal antibody therapy. All three interferon preparations have direct in vitro antigrowth activity for 38C13 and SL2. All three interferons have direct antitumor activity in vivo for 38C13 lymphoma at high doses; however, none of these interferons has independent antitumor activity for SL2 in vivo. These data indicate that there is no relationship between in vitro growth cytostasis/cytolysis and in vivo antitumor response. All three interferon preparations will potentiate both tumor specific and nonspecific monoclonal antibody therapy. Natural MIFN-alpha/beta and recombinant human hybrid interferon-alpha A/D, which should share a common cell surface receptor, had similar antitumor activity in both models. Combining recombinant human hybrid interferon-alpha A/D and rMIFN-gamma therapy was not additive for 38C13 lymphoma and a three-way combination with antiidiotype was not significantly more effective than combination therapy with one interferon type. In general, rMIFN-gamma was more effective in in vivo combination therapy against the s.c. T-cell lymphoma than against the i.p. B-cell lymphoma and was more synergistic with anti-Thy1.1 than with antiidiotype.

Abstract

mAb directed toward the idiotype of the 38C13 murine B cell lymphoma can be used to treat and cure a high percentage of mice challenged previously with an otherwise lethal dose of tumor cells. Tumors developing in animals despite antibody therapy were examined by immunofluorescence and found to demonstrate either loss of surface Ig, or expression of an altered idiotype that no longer bound the antibody used for treatment. Further immunofluorescence analysis of the variant tumors revealed individual patterns of cross-reactivity with anti-38C13 idiotype mAb other than that used for therapy. The variant tumor cells were fused to myeloma cells and hybrids were isolated which secreted large quantities of the altered idiotype proteins. Polyclonal antibodies and mAb prepared against the mutant proteins demonstrated cross-reactivity with the original 38C13 protein and its other variants. But the variants and wild type cells could be distinguished from each other by their patterns of reactivity with the panels of anti-idiotype antibodies. Differences in apparent m.w. were demonstrated in the L chains of each of the mutant proteins. Southern blot analysis of the H chain locus of these mutants established that they were all clonally related; however, the L chain loci were grossly different. Thus, rare cells with alteration in their Ig L chain genes and expressed proteins can give rise to idiotype variants in this B cell tumor.

Abstract

We have previously reported on the augmentation of monoclonal anti-Id antibodies by IL-2 in the therapy of a murine B cell lymphoma. The mechanism of this augmentation was through the expansion by IL-2 of effector cells mediating antibody-dependent cellular cytotoxicity. In this paper we explore the power of IL-2 to enhance anti-Id therapy on larger tumor burdens and at sites distant from the site of injection. The combination treatment was able to induce regression of established 1-cm s.c. tumors associated with microscopic metastatic tumor in lungs, liver, and spleen. Further studies into the mechanism of activity showed that IL-2 was unable to augment in vivo or in vitro tumor lysis by F(ab')2 fragments, thus emphasizing the importance of Fc interactions with antibody-dependent cellular cytotoxicity effector cells. FcR-bearing NK cells were increased in the peritoneum of IL-2-treated mice. The augmented therapeutic effect by the combination treatment was specific for tumor cells expressing the target Id, and non-specific cytotoxicity on Id-negative variants was not seen.

Abstract

Immunoglobulin (Ig) or idiotype (Id) is a tumor-specific target in those B cell malignancies that express this molecule on their surface. We explored the biology of B cell acute lymphoblastic leukemia (B cell ALL) using Id as a tumor marker. In this report we describe the development of anti-Id monoclonal antibodies (MAB) for two children with B cell ALL. These reagents were used retrospectively to study tumor kinetics and to detect residual disease after chemotherapy. In both cases serum Id values were strikingly high at diagnosis (1.2 mg/mL and 10.8 mg/mL), suggesting that the tumor cells were relatively mature B cells capable of significant antibody production. In both patients the serum Id levels fell with the institution of therapy and confirmed that the patients were in remission. Increasing serum Id predicted relapse four months before conventional methods in patient 1, and Id proved to be a more sensitive measure of tumor burden than Southern blot analysis of rearranged Ig genes in bone marrow samples. Surprisingly, low levels of Id were redetected in the second patient just before completing therapy and have persisted for over a year despite the absence of clinical evidence of recurrent disease. Thus, serum Id levels reflect tumor burden during initial therapy but may not necessarily predict tumor progression after a complete clinical remission.

Abstract

To investigate the possible relatedness of the subpopulations that make up so-called biclonal lymphomas, we examined five bigenotypic and biphenotypic follicular lymphomas using DNA probes specific for the t(14;18) chromosomal translocation, which is a characteristic feature of these neoplasms. On Southern blot analysis, both subpopulations from four of five lymphomas contained comigrating t(14;18) DNA rearrangements, confirming the single cell origins for these neoplasms. No comigrating t(14;18) DNA rearrangements were observed in the fifth lymphoma, but nucleotide sequence analysis of cloned, breakpoint DNA showed identical t(14;18) crossovers in the two subpopulations. The migration differences of both the Ig and chromosome 18 DNA rearrangements were shown to result from somatically acquired mutations of the Ig genes from the fifth lymphoma. These studies indicate that Ig gene rearrangements and idiotope expression are not consistently stable clonal markers since they are subject to variability as a result of somatic mutation. Although translocated chromosome 18 DNA rearrangements are more reliable, they may also vary among cells of some tumors since somatic mutation can affect, as well, DNA of translocated alleles in follicular lymphomas.

Abstract

Small unilamellar liposomes containing methotrexate or methotrexate-gamma-aspartate were conjugated to Staphylococcus aureus protein A and were thus able to bind cell-specific immunoglobulins for targeting to malignant human B- and T-cell lines. We were able to demonstrate enhanced protein A liposome uptake and growth inhibition by targeting with an anti-major histocompatibility complex class II antibody recognizing two different B-cell lines. The enhanced growth inhibition was specific for the targeting antibody and amounted to a 2- to 3-fold lowering of the concentration of drug required to inhibit cell growth by 50% as compared to nontargeted liposomes or liposomes targeted with an antibody not recognizing a cell surface antigen. A strong association between enhanced growth inhibition and liposome internalization as assessed by fluorescent-activated cell sorter analysis of carboxyfluorescein containing protein A liposomes was seen. By contrast, specific enhancement of growth inhibition was not seen with several anti-idiotype antibodies or antibodies to T-cell differentiation antigens. Liposome internalization did not occur with these antibodies. Failure of growth inhibition and PA liposome internalization could not be explained by differences in cell binding of the antibody PA liposomes or the degree of protein A binding of the targeting antibody. Although the ability of the targeting antibody to bind to the cell and to protein A are important, these factors alone are not sufficient to guarantee internalization and growth inhibition. Variations in rates of internalization of various cell surface antigen-antibody complexes may account for different protein A liposome mediated cytotoxicities.

Abstract

C3H/HeN mice were immunized with idiotypic immunoglobulin M (IgM) and its molecular subunits from the syngeneic 38C13 lymphoma. Immunization with idiotypic IgM (38C-Id) resulted in idiotype-specific humoral and cellular immunity and protection against a lethal tumor cell challenge. Heavy (H38C) and light (L38C) chains were isolated by electroelution from preparative polyacrylamide gels. Both of these immunogens induced significant resistance to a subsequent tumor challenge. Variable region immunogens, in the form of trpE-fusion proteins, were obtained by cloning heavy and light chain variable region genes into the expression plasmid pATH-11. Of these, only the trpE-VH38C immunogen yielded immune resistance to tumor challenge. Finally, the nucleic acid sequence of 38C-Id light chain was determined and, based on the corresponding amino acid sequence and an analysis of predicted secondary structure, a region of potential antigenicity in complementarity-determining region 3 was chosen for the production of a synthetic peptide. Vaccination with this synthetic peptide resulted in significant suppression of tumor growth. Analysis of the humoral and cellular immunity generated by these vaccines revealed the presence of antibodies reactive with native idiotypic IgM only in 38C-Id, H38C, and trpE-VH38C immune sera, although the latter two were not idiotype-specific. Idiotype-specific lymphocytes, which proliferated in response to native 38C-Id, were observed in all immune animals. With the exception of the fusion protein immunogens, conjugation to an immunogenic carrier protein (keyhole limpet hemocyanin or thyroglobulin) was required for optimal humoral and cellular responses.

Abstract

A transplantable murine B cell lymphoma was used to study combination therapy with anti-idiotype antibody and interleukin 2 (IL 2). Class-switched IgG2a and IgG2b antibodies were compared. A marked additive and sometimes synergistic effect was seen when IL 2 was combined with either IgG2a or IgG2b anti-idiotype antibodies. A synergistic effect was also seen when similar experiments were performed in nude mice. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays showed that IL 2 enhanced antibody-mediated lysis by peritoneal cells exposed to IL 2 in vitro in a dose-related manner. Peritoneal cells harvested from mice treated in vivo with IL 2 contained an increased number of T cells and asialo GM+ natural killer cells, and also mediated enhanced ADCC. Depletion of natural killer cells with anti-asialo GM and complement resulted in a marked decrease in the antibody-dependent cytotoxicity mediated by these peritoneal cells. The mechanism of synergy between monoclonal antibody and IL 2 may be due to the direct or indirect activation of natural killer cells mediating ADCC.

Abstract

A panel of anti-idiotypic antibodies to the T cell line HPB-ALL was produced by screening with a novel enzyme-linked immunoadsorption assay (ELISA). Using the beta framework I (beta F1) monoclonal antibody directed at a common determinant on the human T cell receptor beta subunit, we were able to specifically capture the receptor molecule from a cell lysate preparation and use this as the basis of an ELISA assay. Hybridoma supernatants were tested for their ability to bind to the receptor thus captured. A total of four antibodies were isolated by this method, and they were shown to immunoprecipitate a disulfide-linked heterodimer composed of alpha (49 kDa) and beta (40 kDa) subunits from HPB-ALL cells, similar to the subunits recognized by the beta F1 antibody. Furthermore, all four antibodies blocked the binding of T40/25, an anti-idiotype to HPB-ALL. Three of these antibodies blocked the binding of anti-Leu 4 to a similar degree as did T40/25, while one did not. This suggests that these new anti-idiotypic antibodies recognize distinct but associated idiotypic determinants. The isolation of such antibodies for any particular T cell line or tumor promises to be useful for biological studies of T cell malignancy in humans.

Abstract

The presence of circulating tumor idiotype interferes with the in vivo effectiveness of anti-idiotype antibodies. We developed two assays that permit identification of patients with high levels of serum idiotype without the need for first producing an anti-idiotype antibody. A cell suspension made from the tumor was cultured for seven days with or without phytohemagglutin (PHA) and/or phorbol myristic acetate (PMA). Ig secretion in vitro by patients' tumor cells varied. In 4 patients, no secretion in vitro occurred, 5 patients had low levels, and 5 patients had high levels of Ig secretion. In three patients, Ig secretion occurred only after stimulation with PHA, PMA, or both. Spontaneous or induced immunoglobulin secretion in vitro is related to the levels of tumor idiotype secretion that exist in vivo. Eight patients with serum idiotype levels greater than 100 micrograms/mL (mean 265 micrograms/mL), had a minimum of 1.0 microgram/10(6) cells of idiotype secretion in vitro. Nine patients with serum idiotype levels less than 30 micrograms/mL (mean 3.7 micrograms/mL), had less than or equal to 0.5 microgram/10(6) cells of idiotype secretion in vitro. In another assay, the levels of IgM kappa and IgM lambda in patients' sera were compared with those in normal serum. An imbalance in the relative amounts of IgM kappa and IgM lambda indicated high levels of circulating idiotype in the serum, but this assay was less sensitive than the in vitro secretion assay and limited to IgM-secreting tumors. These assays will be useful for future clinical studies using anti-idiotype antibodies.

Abstract

Monoclonal anti-idiotype antibodies can be made which are exquisitely specific for B lymphocytic malignancies. We have conducted a clinical trial in which some patients' tumors regressed after infusion of such antibodies. Here, we evaluated characteristics of the antibodies, the tumors, and the patients to determine which features best correlated with the clinical response. Neither the isotype of the murine antibodies, nor their avidity were predictive of clinical outcome. The specific epitope to which the antibodies bound was characterized by immunochemical techniques. Reactivity with a heavy-light chain combinatorial determinant correlated somewhat with clinical effect. Variations in the characteristics of the individual tumors such as antigen sites per cell and ability to modulate the surface immunoglobulin were not predictive of response. In one patient with prolymphocytic leukemia the anti-idiotype antibody had a direct antiproliferative effect on tumor cells in vitro. This patient's tumor response was explainable by such a direct mechanism. In the other patients, who had lymphomas, therapeutic outcome correlated with the number of host nontumor cells infiltrating the tumor. The vast majority of these nontumor cells were mature T lymphocytes of the Leu 4, Leu 3 (T3, T4) phenotype. Thus, a preexistent host-tumor interaction seems to be important in the in vivo effect of anti-idiotype antibodies in B cell tumors.

Abstract

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.

Abstract

A novel 42,000 dalton antigen (MB-1) expressed by mature human B cells in blood and tonsil was identified and characterized by utilizing a hybridoma monoclonal antibody. A comparison of MB-1 with other known B cell antigens suggests that the MB-1 antigen has not been previously identified. From one-and two-color immunofluorescence studies, it appears that the MB-1 antigen is found on all normal immunoglobulin (Ig)-expressing cells, but not on T cells, thymocytes, granulocytes, or platelets. Studies of malignant B cell tumors reveal that the antigen is expressed by virtually all Ig-expressing B cell tumors but only 10% of SIg- B-lineage leukemias. Data from these studies suggest that the MB-1 antigen is expressed late in B cell ontogeny before the expression of SIg.

Abstract

Surface idiotype (Id) of B cell malignancies is an excellent tumor-specific marker. We have, however, recently described heterogeneity of tumor Id in some cases. We therefore sought a way to isolate, reliably and efficiently, different species of idiotype from a potentially heterogeneous population. In this report we demonstrate our success using a series of mouse X human heterohybridomas as fusion partners with human B cell tumors. Three lines (K6H6/B5, K6H9/G12, SBC/H20) demonstrated excellent fusion efficiency with 75%-85% of wells plated containing hybrids. Two cell lines, K6H9/G12 and SBC/H20 had a tendency to secrete a single Ig chain (heavy or light chain), whereas the K6H6/B5 cell line secreted whole immunoglobulin (Ig) in greater than 80% of the hybrids. This line secreted significant amounts of Ig (2.73 micrograms/ml/10(6) cells) and was relatively stable in culture. Since this line has such a high fusion efficiency the products of normal B cells admixed with tumor may be recovered, allowing the opportunity of isolating host anti-tumor antibodies. In order to prove that hybrids were derived from the tumor, Southern blot analysis of rearranged DNA was performed in selected cases. Fusions with this line provide the potential for recovering many different species of idiotype in a mixed population. This will facilitate the production of mouse monoclonal anti-idiotype antibodies against many variants and against different idiotopes.

Abstract

Following treatment of a human B cell lymphoma with an anti-idiotype antibody, a subpopulation of tumor cells remained that had lost the tumor-specific heavy chain idiotypic determinant. Nucleotide sequence analyses of eight independent heavy chain variable region isolates showed extensive point mutations, so that no two sequences were identical. Comparison of pretreatment and posttreatment sequences implicated an amino acid in CDR2 as being involved in the idiotypic determinant. Apparently the malignant B cells escaped the therapeutic effects of the anti-idiotype antibody through an ongoing process of somatic mutation in their immunoglobulin genes. Non-random clustering of amino acid replacements in CDR2 suggested that growth of the tumor may have been influenced by endogenous selective forces interacting with the tumor cell-surface immunoglobulin.

Abstract

The nucleic acid sequence of the heavy chain variable region (VH) expressed by 38C13, a B cell tumor of C3H origin, was determined by a combination of direct (messenger RNA) mRNA sequencing by primer extension and complementary DNA (cDNA) isolation and sequencing in M13. The VH amino acid sequence was deduced, and hypervariable regions were identified. From an analysis of predicted secondary structure, regions of predicted antigenicity were chosen, and a series of synthetic peptides corresponding to CDR2 and CDR3 (complementarity-determining region) were produced. These peptides were coupled to protein carriers and used to immunize syngeneic C3H mice. All peptides gave rise to a vigorous antibody response. However, only the CDR3 peptides induced antibodies that crossreacted with the isolated H chain protein. Only one CDR3 peptide induced antibody-producing clones, isolated as hybridomas, that reacted with the intact IgM protein. However, the appearance of these clones was a low-frequency event. All antibodies reacting with the H chain or the intact IgM protein were idiotypically specific for 38C13. These monoclonal antiidiotype (anti-Id) antibodies, raised against CDR3 peptides, gave strong reactions in enzyme-linked immunosorbent assays and immunoblots, but they were of low affinity compared to syngeneic anti-Id raised against the intact IgM protein. Moreover, while the intact IgM was capable of inducing tumor immunity, the CDR peptides were not able to do so.

Abstract

Monoclonal antibodies which bind to tumour cell surface antigens have produced regressions of malignancies in an increasing number of clinical trials. The largest experience to date is in the treatment of refractory B and T lymphoid tumours using a variety of intravenously administered mouse monoclonal antibodies. Treatment with antibodies against common differentiation antigens or very specific anti-idiotype antibodies has been effective in both cases. Toxicity has been acceptably low. A number of problems which limit the application and efficacy of monoclonal antibody therapy of lymphoid malignancy have been identified. Most prominent among these are tumour heterogeneity, which allows non-antibody binding subpopulations of the tumour to escape therapy, and the patient's immunological response to the monoclonal antibody-tumour cell complex. As more experience is accumulated, solutions to these problems will be found.

Abstract

We studied two patients with malignant B-cell lymphoma that manifested resistance to the therapeutic effects of anti-idiotype antibody because of the emergence of subclones with changes in their immunoglobulin idiotypes. In both patients, tumor-cell populations arose that were unreactive with anti-idiotype antibody but that retained surface immunoglobulin. One of the patients had an additional subpopulation of tumor cells that had switched from mu to gamma heavy-chain expression. Study of the immunoglobulin genes in the tumors confirmed that the subpopulations were derived from the same original clone of neoplastic B cells in each patient. The available data suggest that the idiotypic variation observed was the result of somatic mutation in the variable region of the active immunoglobulin genes. The fact that such mutations became evident over a short time and in the context of a partial tumor response suggests that the antibody therapy exerted a strong selective force against tumor cells that expressed the idiotype determinant. Multiple anti-idiotype antibodies may therefore be needed to identify all cells of a malignant clone, and some patients may require treatment with more than one monoclonal antibody.

Abstract

Eleven patients with B lymphocytic malignancy were treated with mouse monoclonal anti-idiotype antibodies. All but one of the patients in this study had received extensive prior treatment with conventional lymphoma therapy. All antibodies were prepared against, and uniquely reactive with, the patient's own tumor. Ten patients were treated with a single antibody, but one patient received three antibodies concurrently. The treatment protocol initially used an escalating dose schedule that was intended to evaluate toxicity, pharmacokinetics and, eventually, to achieve appreciable levels of free mouse antibody in the circulation. The last two patients received substantial initial doses. Tumor sampling was performed before and during therapy to evaluate tissue penetration by antibody. None of the patients had serum paraproteins by routine clinical testing, but six had idiotype protein detectable by a sensitive immunoassay at levels greater than 1 microgram/mL, two of which were greater than 200 micrograms/mL. Plasmapheresis was capable of reducing these levels temporarily. However, the presence of serum idiotype increased the requirement for mouse antibody to achieve tumor penetration. Another obstacle to treatment was immune response to mouse Ig, which occurred in five of the 11 patients. Once an immune response had begun, further infusions of antibody were not capable of reaching the tumor or inducing tumor regression and were associated with toxicity. Our initial patient remains in an unmaintained complete remission 42 months after receiving antibody. Five of ten additional patients have had objective remissions that were also clinically significant. However, these remissions were not complete and were of relatively short duration. This therapy shows promise as an alternative modality for the treatment of B cell malignancy. Further study will be needed to determine the mechanisms of the antitumor effect and to improve the clinical results.

Abstract

To correlate treatment responses with numbers and types of "host cell infiltrates," lymphoid tissues from 10 patients with low-grade B cell malignancies were stained before, during, and after anti-idiotype therapy with a panel of monoclonal antibodies applied to frozen sections. Tissue penetration by the anti-idiotype antibodies was confirmed in five patients by these immunoperoxidase methods. Large numbers of phenotypic T helper cells were the main component of the "host infiltrate" in most patients. Two patients showed a complete and a near-complete clinical remission, four others had partial responses, and four did not respond to therapy. The two patients that developed clinical remission demonstrated the largest number of T cells, T helper cells, TAC+ cells, Leu-7+ cells, and in general the smallest number of proliferating cells as measured by the Ki-67 antibody. Other major differences in host cells were not evident among the patients. These preliminary data suggest that the type and amount of "host infiltrate" in low-grade B cell lymphomas may predict which patients will respond to anti-idiotype therapy.

Abstract

As an alternative to morphological identification of circulating neoplastic lymphocytes in the blood of patients with non-Hodgkin's lymphoma, DNA of peripheral blood lymphocytes was analysed for clonal immunoglobulin gene rearrangements in 29 patients with low-grade B-cell lymphomas. 76% of the patients showed clonal rearrangements of immunoglobulin genes in their blood, including 58% of those with no other evidence of disease. In seven patients from whom paired samples were available the rearranged bands found in the blood and in lymph-node biopsy specimens containing histologically confirmed lymphoma were identical. Detection of circulating lymphoma cells by use of tumour-specific anti-idiotype antibodies and cytofluorimetry showed complete agreement with the results of DNA analysis.

Abstract

The cells of most tumors are considered to be genetically homogeneous because they are assumed to represent a single clone descended from one abnormal cell. We have discovered three cases of B-cell lymphoma for which this generalization is not true. In each case, the tumor was composed of two subpopulations of cells, each expressing a different immunoglobulin molecule. Antibodies directed against these immunoglobulins were used to separate the two cell subpopulations of each tumor on a fluorescence-activated cell sorter. DNA extracted from the original tumor and the two fractionated subpopulations was analyzed to determine the configuration of immunoglobulin genes. Differences were found in the arrangement of DNA in at least one immunoglobulin gene for each of the two subpopulations. Thus, biclonality of these tumors was revealed by examination of both protein markers (cell-surface immunoglobulin) and DNA markers (immunoglobulin-gene rearrangements). Our results indicate that the incidence of biclonal B-cell lymphoma may be higher than previously recognized, possibly as high as 10 per cent of all B-cell lymphomas. Furthermore, our findings may have important implications for the diagnosis and therapy of lymphoid cancers.

Abstract

Ninety-five diffuse large cell lymphomas in 89 patients were stained in cryostat sections with a panel of monoclonal antibodies. Lymphoma cells from 47 patients (53%) expressed either kappa or lambda light chains, usually in combination with mu heavy chains. Fifteen samples from 12 patients (14%) expressed two or more T-cell antigens and commonly expressed Ia antigens. Lymphoma cells from 10 of these patients uniformly lacked one or more pan T-cell antigens; lymphoma cells from 4 of these patients also lacked both T-subset antigens--findings which should prove useful in diagnosis. Lymphomas from 28 patients (31%) did not express immunoglobulin or T-cell antigens but commonly expressed the B-lineage antigen B1; and the remaining 9 cases generally expressed Ia antigens, common ALL antigens, or both. Our findings confirm the marked immunologic heterogeneity of diffuse large cell lymphomas; the phenotypic heterogeneity observed in T-cell cases in many instances is difficult to reconcile with current models of T-cell differentiation.

Abstract

The immunologic phenotypes of 78 diffuse large cell lymphomas were determined by an immunoperoxidase technique using a panel of monoclonal antibodies. The phenotypes were correlated with clinical and morphological parameters by univariate and multivariate analysis. Forty-one lymphomas (53%) expressed immunoglobulin (Ig+). Of the 37 cases that did not express immunoglobulin (Ig-), 9 expressed T cell antigens. Although the T cell phenotypes were antigenically heterogeneous, all cases represented mature T cell phenotypes. The majority of the remaining 28 cases expressed the B cell-associated antigen, B1. At 5 yr, actuarial survival for the Ig- patients was 63%, compared with 15% for the Ig+ patients. A significantly greater proportion of patients with Ig+ lymphomas were over the age of 65 at diagnosis. All of the 9 patients with marrow involvement were Ig+. Multiple factors were analyzed by the Cox regression procedure for their impact on survival, including antigenic profile, histologic grade, morphological classification, and numerous clinical parameters previously recognized to be of prognostic significance. In this analysis, stage, age greater than 65 yr, systemic symptoms, and marrow involvement had the greatest influence on survival. The survival difference between Ig- and Ig+ patients is explained by a higher proportion of Ig+ patients with these unfavorable prognostic factors. With our current immunologic methods, retrospective cell phenotyping analysis has not provided independent prognostic significance in diffuse large cell lymphoma. A prospective evaluation of similarly treated patients is needed to characterize the influence of phenotype fully and to determine its potential usefulness for therapy.

Abstract

In this report, the authors describe a murine anti-human monoclonal antibody, L3B12, which defines a pan-leukocyte cell surface antigen of approximately 180,000 m.w. Extensive screening against a variety of tissues indicates that L3B12 is sensitive and specific for leukocytes, related cells of bone marrow lineage, and their corresponding neoplasms. Unlike many lymphoid antigens that are not detectable following routine fixation and embedding, those recognized by L3B12 and related antibodies are variably preserved. L3B12 has proven useful in studying the antigen expression of normal leukocytic elements, lymphomas, and related disorders, and in enriching or depleting leukocytes from heterogeneous cell populations. From a diagnostic standpoint, L3B12 staining of tissue sections or cell suspensions is useful for distinguishing large cell lymphomas from undifferentiated carcinomas and in distinguishing lymphomas and leukemias from other small round cell tumors of childhood.

Abstract

We produced a hybridoma designated 4G7 from a mouse immunized with chronic lymphocytic leukemia cells. The 4G7 hybridoma secretes an IgG1 antibody that is specific for normal and malignant B lymphocytes. Using dual color immunofluorescence staining, this antibody reacted with all immunoglobulin-positive cells but no T cells in normal peripheral blood. There was no detectable 4G7 antigen on monocytes, platelets, red cells, granulocytes, or phytohemagglutinin-activated T cells. When PBL were depleted of 4G7 positive cells and stimulated with pokeweed mitogen, secreted immunoglobulin levels fell to less than 10% of control values on Day 5 and less than 1% of control on Day 7. This antibody was reactive with 155 of 176 B lineage neoplasms on which it was screened. Thirty-five cases of myeloid or T-lymphoid malignancy were negative. Our studies show that the 4G7 antigen modulates in the presence of excess antibody. Free 4G7 antigen was not found circulating in human serum. The cell surface antigen identified by 4G7 was sensitive to pronase proteolysis but resistant to trypsin and chymotrypsin digestion. A comparison of 4G7 with other known B-cell antibodies indicates that the 4G7 antigen has not been previously identified. This antibody is of use for the identification of normal B lymphocytes, the study of B-cell differentiation, and the characterization of lymphoid malignancies.

Abstract

Murine monoclonal antibodies (MAB) against the idiotype (Id) of B lymphocyte malignancies are powerful reagents for the study of these diseases, and are potentially useful for treatment. Different strategies for the production of these anti-Id MAB have been compared. Initially, the Id Ig from nonsecreting B cell tumors was "rescued" by human X mouse or human X human hybridization. These somatic cell hybridizations resulted in the secretion of human Ig in 10 and 100% of the fusions, respectively. In a second step, anti-Id MAB were produced by using the "rescued" Id Ig as immunogen. A more streamlined approach is based on a one-step procedure in which the tumor cell suspension is used as immunogen. This method of immunization, coupled with a four-layer ELISA, results in the detection of anti-Id MAB in a frequency of approximately 1% of the total hybrids. By using a pool of 10 different anti-Id MAB, each reactive with the tumor of one patient, we searched for idiotypic relatedness among a panel of 50 additional tumors. No cross-reactions were found, indicating that our current strategy results in the identification of unique idiotypic determinants among human B cell tumors. Idiotypic Ig can be found in the serum of patients with B cell tumors. Among groups of patients, there is a wide spectrum of serum Id levels, ranging from less than 0.01 microgram/ml to greater than 500 micrograms/ml.

Abstract

Immunophenotyping studies with monoclonal antibodies have revealed the heterogeneity of childhood acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). The lymphoid malignancies of T-cell lineage are particularly heterogeneous and, until now, no single monoclonal antibody has been found to identify all cases of T-ALL and T-NHL. A monoclonal antibody, 4H9, recognizes an antigen of 40,000 molecular weight on normal and malignant T cells. Thirty-six cases of childhood T-ALL and T-NHL were tested, and in all cases, the malignant blast cells were reactive with 4H9, whereas malignant cells from 61 cases of non-T ALL and NHL were not reactive with 4H9. Monoclonal antibody 4H9 is a sensitive and specific reagent for the identification of childhood T-cell ALL and NHL and should be extremely useful in immunophenotyping studies of lymphoid malignancies.

STUDIES OF A HUMAN LYMPHOCYTE-T ANTIGEN RECOGNIZED BY A MONOCLONAL-ANTIBODYPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESEngleman, E. G., Warnke, R., FOX, R. I., Dilley, J., Benike, C. J., Levy, R.1981; 78 (3): 1791-1795

Abstract

A monoclonal antibody (designated L17F12) detects an antigen present on 95-100% of human peripheral T lymphocytes, the majority of thymocytes, and acute lymphocytic leukemia T cells but not B cells, B-cell lines, or monocytes. Examination of frozen tissue sections by the immunoperoxidase method revealed that the cells expressing this antigen were found predominantly in the medulla of thymus and in T-cell zones of lymph node and spleen. The antigen recognized by L17F12 was associated with a cell-surface glycoprotein of 67,000 daltons. L17F12 was used to isolate this molecule from human thymocytes, normal peripheral T cells, leukemic T cells, and T-cell lines. Expression of this antigen on normal T cells was not diminished by prolonged exposure in vitro to various T-cell stimuli. In the absence of complement, L17F12 bound to T cells without altering proliferative functions, thus enabling rapid purification of functionally intact T cells. In the presence of complement, L17F12 was cytolytic for T cells, providing the basis for depletion of T cells from heterogeneous populations. These data suggest that the monoclonal antibody L17F12 recognizes a specific T-cell differentiation protein. This antibody will be useful in studies of the human immune system.

Abstract

A set of stable mouse-human hybrids was constructed from the neoplastic lymphocytes from a patient with nodular lymphoma and from another with chronic lymphocytic leukaemia. Both patients had shown a clinical response to human leucocyte interferon. The same interferon preparation inhibited the growth rate of 14 out of 17 established hybrid cell lines. This system provides evidence of a direct growth inhibitory effect of interferon on neoplastic B lymphocytes. Such a system could be used to predict the sensitivity of a patient's tumour before therapy.

HUMAN THYMUS-LEUKEMIA ANTIGEN DEFINED BY HYBRIDOMA MONOCLONAL-ANTIBODIESPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALevy, R., Dilley, J., FOX, R. I., Warnke, R.1979; 76 (12): 6552-6556

Abstract

A series of mouse hybridomas producing monoclonal antibodies against human acute lymphocytic leukemia (ALL) cells was generated and screened for tumor specificity. Among 1200 primary cultures, 60 produced an antibody that could distinguish between the immunizing leukemia cells and an isologous B lymphoblastoid cell line. Of these, two produced an antibody that detects an antigen expressed preferentially on ALL cells and on a subpopulation of normal cells found in the cortex of the thymus. Other normal human lymphoid cells from lymph nodes, spleen, bone marrow, and peripheral blood express only low levels of this antigen. High levels of this "thymus-leukemia" antigen were found on T-ALL cells, T-ALL-derived cell lines, and some "null" ALL cells. By contrast, B-cell leukemias, B lymphoblastoid cell lines, and normal and malignant myeloid cells contain either low or undetectable amounts of this antigen. The thymus-leukemia antigen has been isolated from the membranes of leukemia cells by detergent solubilization and subsequent immunoprecipitation with the monoclonal antibody. Preliminary biochemical characterization shows the antigen to be associated with a polypeptide of Mr approximately 28,000.